Nitrate Starvation Induces Lateral Root Organogenesis in Triticum aestivum via Auxin Signaling

Abstract

The lateral root (LR) is an essential component of the plant root system, performing important functions for nutrient and water uptake in plants and playing a pivotal role in cereal crop productivity. Nitrate (NO₃^-) is an essential nutrient for plants. In this study, wheat plants were grown in 1/2 strength Hoagland's solution containing 5 mM NO₃^- (check; CK), 0.1 mM NO₃^- (low NO₃^-; LN), or 0.1 mM NO₃^- plus 60 mg/L 2,3,5-triiodobenzoic acid (TIBA) (LNT). The results showed that LN increased the LR number significantly at 48 h after treatment compared with CK, while not increasing the root biomass, and LNT significantly decreased the LR number and root biomass. The transcriptomic analysis showed that LN induced the expression of genes related to root IAA synthesis and transport and cell wall remodeling, and it was suppressed in the LNT conditions. A physiological assay revealed that the LN conditions increased the activity of IAA biosynthesis-related enzymes, the concentrations of tryptophan and IAA, and the activity of cell wall remodeling enzymes in the roots, whereas the content of polysaccharides in the LRP cell wall was significantly decreased compared with the control. Fourier-transform infrared spectroscopy and atomic microscopy revealed that the content of cell wall polysaccharides decreased and the cell wall elasticity of LR primordia (LRP) increased under the LN conditions. The effects of LN on IAA synthesis and polar transport, cell wall remodeling, and LR development were abolished when TIBA was applied. Our findings indicate that NO₃^- starvation may improve auxin homeostasis and the biological properties of the LRP cell wall and thus promote LR initiation, while TIBA addition dampens the effects of LN on auxin signaling, gene expression, physiological processes, and the root architecture.

Keywords: cell wall; indole-3-acetic acid; lateral root; nitrate; wheat.

Figure 1

Net increases in dry weight of shoots (A) and roots (B) of wheat seedlings grown in solution containing 5 mM NO₃⁻ (CK), 0.1 mM NO₃⁻ (LN), and 0.1 mM NO₃⁻ + 60 mg/L TIBA (LNT) at 12, 24, and 48 h after treatment. Values represent the mean ± SD of at least three independent experiments. Different letters above columns indicate significant differences at p ≤ 0.05.

Figure 2

Representations of the root morphology of wheat seedlings at 48 h after treatment with 5 mM NO₃⁻ (CK), 0.1 mM NO₃⁻ (LN), and 0.1 mM NO₃⁻ + 60 mg/L TIBA (LNT). Bar = 2 cm.

Figure 3

Paired comparisons of DEGs between different N treatments. Wheat seedlings grown in solution containing 5 mM NO₃⁻ (CK), 0.1 mM NO₃⁻ (LN), and 0.1 mM NO₃⁻ + 60 mg/L TIBA (LNT) at 48 h after treatment.

Figure 4

GO analysis of DEGs for the roots of wheat seedlings treated with 5 mM NO₃⁻ (CK), 0.1 mM NO₃⁻ (LN), and 0.1 mM NO₃⁻ + 60 mg/L TIBA (LNT) for 48 h. (A) LN vs. CK; (B) LNT vs. LN. The X-axis indicates the number of differential genes annotated for the GO count, and the Y-axis indicates the enriched GO terms. The color gradient from red to blue represents the significance level of the −Log₁₀(p-value). The key functions of DEGs that were involved in auxin signaling, cell wall remodeling, nitrogen mentalism, and water transport have been highlighted using the red font.

Figure 5

KEGG analyses of DEGs for the roots of wheat seedlings treated with 5 mM NO₃⁻ (CK), 0.1 mM NO₃⁻ (LN), and 0.1 mM NO₃⁻ + 60 mg/L TIBA (LNT) for 48 h. (A) The KEGG enrichment analysis of LN compared with CK. (B) The KEGG enrichment analysis of LNT compared with LN. The X-axis in the figure is the GeneRatio; the Y-axis is the KEGG pathway. The size of the dots represents the number of genes annotated for the KEGG pathway, and the color gradient from red to green represents the significance level of the enrichment. The key enriched pathways for DEGs that were involved in auxin biosynthesis and transport and nitrogen metabolism were highlighted using the red font.

Figure 6

A schematic model of the Trp-dependent IAA biosynthetic pathway (A) and the expression of DEGs involved in the pathways (B). Wheat seedlings were treated with 5 mM NO₃⁻ (CK), 0.1 mM NO₃⁻ (LN), and 0.1 mM NO₃⁻ + 60 mg/L TIBA (LNT) for 48 h. AO, indole-3-acetaldehyde oxidase; DAO, 2-oxoglutarate-dependent dioxygenase; GH3, indole-3-acetic acid-amido synthetase; IAA-AA, indole-3-acetic acid–amino acid conjugate; IAAId, indole-3-acetaldehyde; IGP, indole-3-glycerophosphate; IGPS, indole-3-glycerol phosphate synthase; ILL, IAA-amino acid hydrolase ILR1-like; IPA, indole-3-pyruvate; TAM, tryptamine; TAR, tryptophan aminotransferase-related protein; TDC1, tryptophan decarboxylase 1; Trp, tryptophan; TSA, tryptophan synthase alpha chain; YUCs, indole-3-pyruvate monooxygenase. Heatmap (B) showing the differential expression levels of the identified DEGs in the roots of wheat plants with different N treatments. The log₂(FoldChange) values for individual genes are indicated by the color, as shown in the scale, with red indicating a high level of expression and blue indicating a low level of expression.

Figure 7

Schematic representation of major auxin transport types (A) and a list of genes related to auxin transport in wheat roots (B). The arrows in (A) indicate auxin flow mediated by a particular transporter. A heatmap (B) showing the differential expression levels of the identified DEGs in the roots with three treatments from three replicates. The color blocks represent log₂-transformed fold changes, with red indicating a high level of expression and blue indicating a low level of expression.

Figure 8

Volcano plots of DEGs involved in cell division, elongation, and the cell cycle. (A) LN vs. CK; (B) LNT vs. LN. Wheat seedlings grown in solution containing 5 mM NO₃^– (CK), 0.1 mM NO₃^– (LN), and 0.1 mM NO₃^– + 60 mg/L TIBA (LNT) at 48 h after treatment. Red and blue dots indicate upregulated and downregulated DEGs, respectively, and gray dots indicate genes that were not differentially expressed.

Figure 9

Volcano plots of DEGs encoding cell wall expansins. (A) LN vs. CK; (B) LNT vs. LN. Wheat seedlings grown in solution containing 5 mM NO₃^– (CK), 0.1 mM NO₃^– (LN), and 0.1 mM NO₃^– + 60 mg/L TIBA (LNT) at 48 h after treatment. Red and blue dots indicate upregulated and downregulated DEGs, respectively, and gray dots indicate genes that were not differentially expressed.

Figure 10

RNA-seq data accuracy verification. The column represents the 2^−ΔΔCt value of qRT-PCR analysis and log₂(FoldChange) value of RNA-Seq. The values represent the mean ± SD. Positive values on the Y-axis indicate the upregulation of hub genes. Wheat seedlings grown in solution containing 5 mM NO₃^– (CK), 0.1 mM NO₃^– (LN), and 0.1 mM NO₃^– + 60 mg/L TIBA (LNT) at 48 h after treatment.

Figure 11

Content of root tryptophan (A) and IAA (B) in wheat seedlings grown under different N treatments. Different letters above columns indicate significant differences at p ≤ 0.05 between treatments. The values represent the mean ± SD of three replicates. Wheat seedlings grown in solution containing 5 mM NO₃^– (CK), 0.1 mM NO₃^– (LN), and 0.1 mM NO₃^– + 60 mg/L TIBA (LNT) at 48 h after treatment.

Figure 12

ATR-FTIR spectra obtained from the epidermal cell walls of LRP of wheat seedlings treated with 5 mM NO₃^– (CK), 0.1 mM NO₃^– (LN), and 0.1 mM NO₃^– + 60 mg/L TIBA (LNT) for 48 h. (A) Difference spectra generated via the digital subtraction of the spectrum of the LN (0.1 mM NO₃^–) LRPs from that of the CK LRPs and the spectrum of the LNT (0.1 mM NO₃^– + 60 mg/L) LRPs from that of the LN LRPs. (B) Graphs show the transmission (Y-axis) plotted against the wavenumber (X-axis).

Figure 13

Graphs representing the LRP elastic moduli of wheat seedlings grown in 5 mM NO₃^– (CK), 0.1 mM NO₃^– (LN), or 0.1 mM NO₃^– + 60 mg/L TIBA (LNT) at 48 h after treatment. Data are the mean ± SD from three LRPs. Different letters indicate statistically significant differences at p < 0.05.