Comparative Proteomic Analysis of Two Contrasting Maize Hybrids' Responses to Low Nitrogen Stress at the Twelve Leaf Stage and Function Verification of ZmTGA Gene

Abstract

Nitrogen is one of the essential nutrients for plant growth and development. However, large amounts of nitrogen fertilizer not only increase the production costs, but also lead to serious environmental problems. Therefore, it is particularly important to reduce the application of nitrogen fertilizer and develop maize varieties with low nitrogen tolerance. The aim of this study was to determine the phenotypic and proteomic alterations of maize affected by nitrogen deficiency and to elucidate the molecular and physiological mechanisms underpinning maize tolerance to low nitrogen. Two maize hybrids with contrasting low nitrogen tolerance were used as the experimental materials. Maize plants were grown under different nitrogen application levels (N0 and N240) and proteomic analysis performed to analyze leaf differentially abundant proteins (DAPs) under different nitrogen conditions. The results showed that under the nitrogen deficiency condition, the nitrogen content, leaf dry weight, leaf area, and leaf area index of XY335 decreased by 15.58%, 8.83%, 3.44%, and 3.44%, respectively. However, in the variety HN138, the same parameters decreased by 56.94%, 11.97%, 8.79%, and 8.79%, respectively. Through proteomic analysis, we found that the low nitrogen tolerance variety responded to low nitrogen stress through lignin biosynthesis, ubiquitin-mediated proteolysis, and stress defense proteins. Transmembrane transporters were differentially expressed in both hybrids after low nitrogen treatment, suggesting that this was a common response to low nitrogen stress. Using bioinformatics analysis, we selected the key candidate gene (ZmTGA) that was assumed to respond to low nitrogen stress, and its function was characterized by maize mutants. The results showed that when compared with normal nitrogen treatment, the root length of the mutants under low nitrogen treatment increased by 10.1%, while that of the wild-type increased by 14.8%; the root surface area of the wild type under low nitrogen treatment increased by 9.6%, while that of the mutants decreased by 5.2%; the root surface area of the wild type was higher than that of the mutant at both nitrogen levels; and the activities of glutathione and guaiacol peroxidase enzymes in the mutant were lower than those in the wild-type under low nitrogen treatment. In summary, the mutant was less adaptable to a low nitrogen environment than the wild type. Our results provide maize genetic resources and a new direction for a further understanding of maize response to low nitrogen stress.

Keywords: DAPs; Zea mays L.; low nitrogen tolerance; morphological characteristics.

Figure 1

Phenotypic and physiological analysis of two maize hybrids under two nitrogen treatments. (A) Leaf nitrogen concentration, (B) leaf dry weight, (C) leaf area, (D) leaf area index. Different letters in the figure indicate significant differences (p < 0.05) between hybrids or treatments.

Figure 2

Protein identification and analysis. (A) Distribution of protein molecular weight, the abscissa is the distribution range of protein molecular weight, and the ordinate is the number of proteins with corresponding molecular weight. (B) Distribution of the protein’s sequence coverage. Each sector represents the proportion of a coverage range. The larger the sector area, the greater the number of proteins with coverage in this range will be. Numbers on the outside of the sector indicate the range of coverage and the proportion of proteins distributed in this region.

Figure 3

Venn diagram analysis of DAPs identified in the four experimental comparisons. Each compared combination is separated by an underscore (e.g., XYT_XYC, the former is divided by the latter).

Figure 4

GO enrichment analysis result diagram of Area I. The horizontal axis represents the GO term, and the vertical axis represents enrichment rate, namely the ratio of the protein number enriched in the GO term to the background number annotated to the GO term. The greater the ratio, the greater the degree of enrichment. The column color gradient indicates the significance of enrichment, where p < 0.05 was marked as *.

Figure 5

Enriched KEGG pathways of Area I. The abscissa represents the pathway name. The ordinate represents the enrichment rate, which refers to the ratio of the number of proteins enriched in this pathway to the number of proteins annotated into the pathway. The higher the ratio, the greater the degree of enrichment. Column color gradient indicates the significance of enrichment, where p < 0.05 is marked with *.

Figure 6

Phenotypic response of maize seedlings under low nitrate (0.04 mM NO³⁻) and optimal nitrate 4 mM NO³⁻) conditions with hydroponics. (A) Mutant tga under normal nitrogen treatment conditions. (B) Mutant tga under low nitrate. (C) Wild-type under normal nitrogen treatment conditions. (D) Wild type under low nitrogen treatment.

Figure 7

Root differences between wild-type TGA and mutant tga at two nitrogen levels. (A) Average root diameter, (B) ratio of fresh weight to root shoot, (C) total root length, (D) root surface area. Different letters indicate significant differences at p < 0.05 between different groups according to the Duncan test. CK, 4 mmol L⁻¹; LN, 0.04 mmol L⁻¹ NO³⁻.

Figure 8

Physiological differences between wild-type TGA and mutant tga at two nitrogen levels. (A) Relative chlorophyll content (SPAD), (B) Glutathione S-transferase (GST) activity, (C) Peroxidase (POD) activity. Different letters indicate significant differences at p < 0.05 between different groups according to the Duncan test. CK, 4 mmol L⁻¹; LN, 0.04 mmol L⁻¹ NO³⁻.