Comparative Proteomic Analysis Unveils Critical Pathways Underlying the Role of Nitrogen Fertilizer Treatment in American Elderberry

Abstract

American elderberry (Sambucus nigra subsp. canadensis) is a rapidly growing specialty crop in Missouri and eastern North America. Nitrogen (N) is a major nutrient involved in plant growth and development. However, proteome changes for different genotypes of elder in response to varying levels of N-treatment remain undefined. To reveal plant responses to N, comparative proteomic analyses were performed to determine consistent changes in three genotypes of elderberry leaves (Adams II, Bob Gordon and Wyldewood) grown under different N-fertilizer treatments. 165 proteins separated by two dimensional gel electrophoresis showed significant differences in abundance (p < 0.05 and greater than 2-fold). Principal component analysis of the abundance profiles of these proteins revealed Bob Gordon as a distinct genotype. The 165 proteins were identified by mass spectrometry and showed similar functional distributions in these genotypes underlying the N-treatment. Among the proteins identified, 23 are mainly involved in photosynthesis, protein metabolism and redox homeostasis. Their abundance profiles were not altered upon exposure to N or genotype. These results provide novel insights into plant responses to fertilizer treatment at the proteome level and could lead to a better understanding of molecular mechanisms of elderberry growth.

Keywords: Sambucus; comparative proteomics; elderberry; nitrogen response.

Figure 1

(a–c) Hierarchical clustering of 165 differentially abundant proteins (p < 0.05) for each N fertilizer concentration (number following A (Adams II), B (Bob Gordon), W (Wyldewood)). The abundance of each protein was transformed using log2. Every colored box represents a protein that is up-regulated (red) or down-regulated (green) in a certain treatment. Gray color means + ∞. Hierarchical clusters (A, B, C, and D) were established as described in the text.

Figure 1

(a–c) Hierarchical clustering of 165 differentially abundant proteins (p < 0.05) for each N fertilizer concentration (number following A (Adams II), B (Bob Gordon), W (Wyldewood)). The abundance of each protein was transformed using log2. Every colored box represents a protein that is up-regulated (red) or down-regulated (green) in a certain treatment. Gray color means + ∞. Hierarchical clusters (A, B, C, and D) were established as described in the text.

Figure 2

The functional category distribution of the 101 identified proteins. Proteomes identified in the leaf were matched to their Arabidopsis orthologues and classified into 16 major functional groups using the Mercator software (gabipd.org/biotools/mercator/). Proteins involved in photosynthesis and protein metabolism were further sub-classified as indicated by arrows. OPP: oxidative pentose phosphate; TCA: tricarboxylic acid; PTM: post translational modification.

Figure 3

Hierarchical clustering of orthologous N characteristic-protein groups consistently enriched in three N groups compared to control (p < 0.05) in different genotypes. The color gradient range of the heat map indicates the proportional up-regulation (red) and down-regulation (green) of protein abundance (fold difference, log2-transformed). Hierarchical clusters (A, B, C, and D) were established as described in the text.

Figure 4

Schematic representation of key pathways that regulate N fertility. These differentially abundant proteins (interaction p < 0.05) across different genotypes and N treatments were also classified in functional categories using MapMan bin codes (http://mapman.gabipd.org). The cellular locations of each protein were determined by the SUBA database (Version 3). Arrows indicate up- and down-regulation of proteins within each set of isoforms compared to control group at a given N condition. Red squares in the functional category indicate an overall regulation of the category at 169 kg N/ha compared to control (0 kg N/ha) in different genotypes, while green squares means down regulation.