Time-Course of Metabolic and Proteomic Responses to Different Nitrate/Ammonium Availabilities in Roots and Leaves of Maize

Abstract

The availability of nitrate and ammonium significantly affects plant growth. Co-provision of both nutrients is generally the best nutritional condition, due to metabolic interactions not yet fully elucidated. In this study, maize grown in hydroponics was exposed to different nitrogen (N) availabilities, consisting of nitrate, ammonium and co-provision. Roots and leaves were analyzed after 6, 30, and 54 h by biochemical evaluations and proteomics. The ammonium-fed plants showed the lowest biomass accumulation and the lowest ratio of inorganic to organic N content, suggesting a metabolic need to assimilate ammonium that was not evident in plants grown in co-provision. The N sources differently affected the root proteome, inducing changes in abundance of proteins involved in N and carbon (C) metabolisms, cell water homeostasis, and cell wall metabolism. Notable among these changes was that some root enzymes, such as asparagine synthetase, phosphoenolpyruvate (PEP) carboxylase, and formate dehydrogenase showed a relevant upsurge only under the sole ammonium nutrition. However, the leaf proteome appeared mainly influenced by total N availability, showing changes in the abundance of several proteins involved in photosynthesis and in energy metabolism. Overall, the study provides novel information about the biochemical determinants involved in plant adaptation to different N mineral forms.

Keywords: ammonium; co-provision; maize; nitrate; plant nutrition; proteomics.

Figure 1

Plant growth evaluated as the fresh biomass of roots (A) and leaves (B) per plant (g FW plant⁻¹). Maize plants were collected at t0 (white bar) or after 6 h (light grey bars), 30 h (grey bars) and 54 h (dark grey bars) of growth in the presence of 5 mM NO₃⁻ (n), 5 mM NH₄⁺ (a) and of 2.5 mM NO₃⁻ + 2.5 mM NH₄⁺ (na). Values are the mean ± standard error (SE) (n = 8). The statistical significance was assessed by analysis of variance (ANOVA) test (p < 0.05, Tukey post hoc method).

Figure 2

Content of NO₃⁻, NH₄⁺ and amino acids in roots and leaves. The graphs report the content of NO₃⁻ in roots (A) and in leaves (B); the content of NH₄⁺ in roots (C) and in leaves (D); the content of amino acids in roots (E) and in leaves (F). Maize plants were collected at t0 (white bar) or after 6 h (light grey bars), 30 h (grey bars), and 54 h (dark grey bars) of growth in presence of 5 mM NO₃⁻ (n), 5 mM NH₄⁺ (a), and 2.5 mM NO₃⁻ + 2.5 mM NH₄⁺ (na). Values are the mean ± SE (n = 3). The statistical significance was assessed by ANOVA test (p < 0.05, Tukey post hoc method).

Figure 3

Contents of sucrose (A,B) and reducing sugars (C,D) in roots (A,C) and leaves (B,D). Maize plants were collected at t0 (white bar) or after 6 h (light grey bars), 30 h (grey bars), or 54 h (dark grey bars) of growth in presence of 5 mM NO₃⁻ (n), 5 mM NH₄⁺ (a) and 2.5 mM NO₃⁻ + 2.5 mM NH₄⁺ (na). Values are the mean ± SE (n = 3). The statistical significance was assessed by ANOVA test (p < 0.05, Tukey post hoc method).

Figure 4

Abundance of the differentially accumulated proteins in maize roots. Maize plants were exposed for 6, 30, and 54 h to the presence of 5 mM NO₃⁻ (n), 5 mM NH₄⁺ (a), and 2.5 mM NO₃⁻ + 2.5 mM NH₄⁺ (na). The image was obtained by means of the PermutMatrix graphical interface after Z-score normalization of the averages of protein Spectrum Intensity % (%SI, n = 3). Each colored cell represents the average of the %SI according to the color scale.

Figure 5

Abundance of the differentially accumulated proteins in maize leaves. Maize plants were exposed for 6, 30, and 54 h to the presence of 5 mM NO₃⁻ (n), 5 mM NH₄⁺ (a), and 2.5 mM NO₃⁻ + 2.5 mM NH₄⁺ (na). The image was obtained by means of the PermutMatrix graphical interface after Z-score normalization of the averages of protein Spectrum Intensity % (%SI, n = 3). Each colored cell represents the average of the %SI according to the color scale.

Figure 6

Functional distribution of the differentially accumulated proteins in maize plants. The proteins differentially accumulated were grouped in classes according to literature and GeneBank. (A) Proteins differentially accumulated in roots; (B) proteins differentially accumulated in leaves. The functional distribution indicates the percentage of each class as compared to the total number of proteins differentially accumulated.

Figure 7

Classification of the differentially accumulated proteins according to the main source of variation in roots (A) and in leaves (B). The proteins differentially accumulated, sorted in functional classes, are categorized in two groups: proteins whose changes were specifically related to the N source (dark grey bars) and proteins whose changes were not related to N sources, but to other factors, such as time and the total N availability (light grey bars).