Insights into nitrogen metabolism in the wild and cultivated lettuce as revealed by transcriptome and weighted gene co-expression network analysis

Abstract

Large amounts of nitrogen fertilizers applied during lettuce (Lactuca sativa L.) production are lost due to leaching or volatilization, causing severe environmental pollution and increased costs of production. Developing lettuce varieties with high nitrogen use efficiency (NUE) is the eco-friendly solution to reduce nitrogen pollution. Hence, in-depth knowledge of nitrogen metabolism and assimilation genes and their regulation is critical for developing high NUE varieties. In this study, we performed comparative transcriptomic analysis of the cultivated lettuce (L. sativa L.) and its wild progenitor (L. serriola) under high and low nitrogen conditions. A total of 2,704 differentially expressed genes were identified. Key enriched biological processes included photosynthesis, oxidation-reduction process, chlorophyll biosynthetic process, and cell redox homeostasis. The transcription factors (TFs) belonging to the ethylene responsive factor family and basic helix-loop-helix family were among the top differentially expressed TFs. Using weighted gene co-expression network analysis we constructed nine co-expression modules. Among these, two modules were further investigated because of their significant association with total nitrogen content and photosynthetic efficiency of photosystem II. Three highly correlated clusters were identified which included hub genes for nitrogen metabolism, secondary metabolites, and carbon assimilation, and were regulated by cluster specific TFs. We found that the expression of nitrogen transportation and assimilation genes varied significantly between the two lettuce species thereby providing the opportunity of introgressing wild alleles into the cultivated germplasm for developing lettuce cultivars with more efficient use of nitrogen.

Figure 1

Phenotypic reaction of two lettuce genotypes to high nitrogen (HN) and low nitrogen (LN) treatments.

Figure 2

Venn diagram showing the number of up- and down regulated genes in Salinas and UC in response to nitrogen stress.

Figure 3

Enriched GO terms according to biological processes (red bars), cellular components (green bars) and molecular functions (orange bars). The x-axis represents log2 of the gene counts.

Figure 4

Differentially expressed transcription factors identified by RNA-Seq analysis.

Figure 5

Mapman analysis of differentially expressed genes related to various metabolic processes in (a) Salinas, (b) UC genotypes. Red squares represent genes that were significantly down-regulated; blue squares represent genes that were significantly up-regulated.

Figure 6

Differential expression of nitrogen metabolism genes Salinas and UC genotypes when grown at high nitrogen (HN) and low nitrogen (LN) treatments.

Figure 7

(a) Gene modules identified by WGCNA. Dendrogram was obtained by clustering dissimilarity with the modules represented by the colored block based on the topological overlap. A total of nine module (colored blocks) were identified where each module represents a set of highly connected genes. (b) Heatmap plot of the gene network. The heatmap depicts Topological Overlap Matrix (TOM) among all genes in the analysis. Light color represents overlap of highly expressed genes and the darker color represent low overlap of genes. Blocks of yellow colors along the diagonal are the modules.

Figure 8

Heat map of relationship between modules and phenotypic traits. The color indicated direction of correlation, values represent Pearson correlation coefficients, and P-values are in parenthesis.

Figure 9

The co-expression network of highly correlated (r² > .90) hub genes and associated TFs identified by applying the Molecular Complex Detection (MCODE) to the networks. (a) The co-expression network of the significant genes in the lightgreen module. (b, c). Two co-expression clusters of the significant genes in the blue module. The TFs in each cluster are red colored.