Integrative Transcriptome and Metabolome Analysis Identifies Potential Pathways Associated with Cadmium Tolerance in Two Maize Inbred Lines

Abstract

Cadmium (Cd) significantly influences the morphological, physiological traits, and transport capacity of plants, but the underlying mechanism of Cd stress still remains to be further studied. In this study, physiological, transcriptomic, and metabolomic analyses were conducted to examine the morphological and physiological traits of two elite maize inbred lines, Chang7_2 (C7_2, a Cd-resistant line) and Zheng58 (Z58, a Cd-sensitive line) under control and Cd stress conditions. The results of morphological traits indicated that C7_2 reduced by 9.50-29.60% under Cd stress, whereas Z58 displayed more pronounced morphological changes ranging from 10.12 to 41.72% under Cd stress. Physiological assessments revealed that C7_2 maintained relatively stable antioxidant enzyme activity, while Z58 demonstrated more rapid alterations in the antioxidant system under Cd stress. Transcriptomic analysis identified 3030 differentially expressed genes (DEGs) unique to C7_2 and 4298 DEGs unique to Z58, with 1746 common DEGs shared between the two lines. Functional annotation revealed that the unique DEGs in C7_2 were mainly enriched in plant hormone signal transduction, plant-pathogen interactions, and the MAPK signaling pathway, while the unique DEGs in Z58 were mainly enriched in ribosome-related functions, plant hormone signal transduction, and phenylpropanoid biosynthesis. Metabolomic analysis identified 12 superclasses encompassing 896 metabolites in C7_2 and Z58, primarily including lipids and lipid-like molecules, organic acids and derivatives, as well as organoheterocyclic compounds. Analysis of differentially accumulated metabolites (DAMs) revealed fewer DAMs were accumulated in C7_2 under Cd stress. Further analysis identified that the three pathways of GPI anchor biosynthesis, glycerophospholipid metabolism, and purine metabolism were among the top 10 metabolic pathways in C7_2 and Z58. The integrative analysis highlighted the crucial roles of phenylpropanoid biosynthesis and zeatin biosynthesis in C7_2 for resistance to Cd stress. This study provides novel insights into the molecular and metabolic pathways underlying Cd tolerance in maize by integrating transcriptomic and metabolomic analyses of two contrasting inbred lines, providing a theoretical foundation for the future breeding of Cd-tolerant varieties.

Keywords: cadmium stress; maize; metabolome; seedling stage; transcriptome.

Figure 1

The morphological changes in C7_2 and Z58 under Cd stress. (A) Morphology of Z58 and C7_2 for 14-day-old seedlings. (B–J) Analysis of morphological traits for C7_2 and Z58. Different characters indicate significant differences at p value < 0.05 (n = 5).

Figure 2

The SOD, POD, and CAT activity and MDA, H₂O₂, and Cd content changes in C7_2 and Z58 under Cd stress. (A) SOD. (B) POD. (C) CAT. (D) MDA. (E) H₂O₂. (F) Cd. The different characters indicate significant differences at p < 0.05 level (n = 5).

Figure 3

Analysis of transcriptome data for C7_2 and Z58 under CK and Cd stress. (A) PCA of the gene expression patterns of C7_2 and Z58 under CK and Cd treatment. The X axis represents PCA1 (83.02% variance), and the Y axis represents PCA2 (10.85% variance). Each sample has three biological duplicates and is represented on the plot by a unique symbol. (B) Heatmap of the gene expression patterns of C7_2 and Z58 under CK and Cd treatment. (C) Venn diagram of the DEGs in C7_2 T vs. C7_2 CK and Z58 T vs. Z58 CK.

Figure 4

GO and KEGG_enrichment analysis for the DEGs of C7_2 and Z58 under Cd stress. (A) GO analysis for the 1746 common DEGs of C7_2 and Z58. (B) KEGG analysis for the 1746 common DEGs of C7_2 and Z58. (C) GO analysis for the 3030 unique DEGs in C7_2. (D) KEGG analysis for the 3030 unique DEGs in C7_2. (E) GO analysis for the 4298 unique DEGs in Z58. (F) KEGG analysis for the 4298 unique DEGs in Z58.

Figure 5

Identification of gene networks related to Cd stress in C7_2 and Z58. (A) Module–trait relationships of modules and the morphological and physiological traits. The horizontal coordinate represents different traits, the vertical coordinate represents different modules, and the filling value represents the correlation size and the statistical test p value. The closer the correlation value is to 1 or −1, the stronger the positive or negative correlation between the module and the samples. The smaller the p value in parentheses, the stronger the significance. (B) Heatmap of the blue and yellow module genes. The upper part is the heat map of gene expression within the modules, and the lower part is the bar plot of the expression of module eigengenes in each sample.

Figure 6

Analysis of metabolome data and metabolite content of C7_2 and Z58. (A) Cluster heat map analysis of DAMs. (B) Principal component analysis (PCA) of DAMs. (C) Differential metabolite content of 12 superclasses under Cd stress. The different characters indicate significant differences at a p < 0.05 level (n = 6).

Figure 7

The Venn and KEGG pathway enrichment analysis for the DAMs of C7_2 and Z58. (A) Venn diagram analysis for the DAMs of C7_2 and Z58. (B) KEGG analysis for DAMs of C7_2 T vs. C7_2 CK. (C) KEGG analysis for DAMs of Z58 T vs. Z58 CK. The horizontal axis represents the enrichment ratio, while the vertical axis represents the enriched pathway name. The color scale indicates different thresholds of the p value, and the size of the dot indicates the number of metabolites corresponding to each pathway.

Figure 8

Analysis of KEGG enrichment pathways for the DEGs and DAMs of C7_2 and Z58. Venn diagram of KEGG enrichment pathways for the DEGs and DAMs of C7_2 (A) and Z58 (B). Bar plot of KEGG enrichment pathways for the DEGs and DAMs of C7_2 (C) and Z58 (D). The dashed blue and orange lines in (C,D) indicate the threshold value of KEGG enrichment pathways for DEGs and DAMs at p value < 0.01 and p value < 0.05, respectively.

Figure 9

Integrated transcriptomic and metabolomic analysis for C7_2 and Z58 in phenylpropanoid biosynthesis and zeatin biosynthesis. (A) DEGs and DAMs of C7_2 and Z58 involved in phenylpropanoid biosynthesis. (B) DEGs and DAMs of C7_2 and Z58 involved in zeatin biosynthesis. The heatmap of (A,B) colored in blue and red indicates gene expression, and the heatmap of (A,B) colored in blue and orange indicates metabolite accumulation.