Transcriptome Sequencing Identified Genes and Gene Ontologies Associated with Early Freezing Tolerance in Maize

Abstract

Originating in a tropical climate, maize has faced great challenges as cultivation has expanded to the majority of the world's temperate zones. In these zones, frost and cold temperatures are major factors that prevent maize from reaching its full yield potential. Among 30 elite maize inbred lines adapted to northern China, we identified two lines of extreme, but opposite, freezing tolerance levels-highly tolerant and highly sensitive. During the seedling stage of these two lines, we used RNA-seq to measure changes in maize whole genome transcriptome before and after freezing treatment. In total, 19,794 genes were expressed, of which 4550 exhibited differential expression due to either treatment (before or after freezing) or line type (tolerant or sensitive). Of the 4550 differently expressed genes, 948 exhibited differential expression due to treatment within line or lines under freezing condition. Analysis of gene ontology found that these 948 genes were significantly enriched for binding functions (DNA binding, ATP binding, and metal ion binding), protein kinase activity, and peptidase activity. Based on their enrichment, literature support, and significant levels of differential expression, 30 of these 948 genes were selected for quantitative real-time PCR (qRT-PCR) validation. The validation confirmed our RNA-Seq-based findings, with squared correlation coefficients of 80% and 50% in the tolerance and sensitive lines, respectively. This study provided valuable resources for further studies to enhance understanding of the molecular mechanisms underlying maize early freezing response and enable targeted breeding strategies for developing varieties with superior frost resistance to achieve yield potential.

Keywords: DEGs; RNA-seq; freezing stress; maize; molecular mechanism.

Figure 1

Seedling performances under freezing treatment. The performances were measured as survival rate and relative electrical conductivity. Survival rates were measured on the third day at 25°C after freezing treatment at −1°C for 3 h for 30 Chinese elite maize inbred lines (A). Seedlings before freezing treatment are demonstrated by the two extreme lines, freezing tolerant KR701 (B) and freezing sensitive Hei8834 (C). Visible differences were observed on the third day of recovery at 25°C after freezing treatment (D) between KR701 (upper row) and Hei8834 (lower row). Relative electrical conductivity was measured for KR701 and Hei8834 before freezing treatment and after 1 h of freezing treatment at −1°C (E). Double asterisks indicate a statistically significant difference (p < 0.01) between KR701 and Hei8834. Mean values are calculated from three biological replicates; error bars represent the standard error of the mean.

Figure 2

Profile of gene expression by inbred line and freezing treatment. The gene expression profile is illustrated as the number of transcriptomic responses by using a Venn diagram. A total of 19,794 genes were expressed. Freezing treatments are labeled Control (C) and Freezing (F). The Tolerant line (KR701) and Sensitive line (Hei8834) are labeled “T” and “S,” respectively. The biological samples of four combinations are CT, FT, CS, and FS, respectively. The area labeled “A” represents the genes specifically expressed in tolerant line KR701 after freezing treatment (FT). The area labeled “B” represents the genes specifically expressed in sensitive line Hei8834 after freezing treatment (FS). The area labeled “C” represents the freezing responsive genes shared by the tolerant and sensitive lines.

Figure 3

Profile of differentially expressed genes (DEGs). The differentiations were compared between inbred lines under each freezing treatment, or between freezing treatments in each inbred line. Freezing treatments are labeled Control (C) and Freezing (F). The tolerant line (KR701) and sensitive line (Hei8834) are labeled “T” and “S,” respectively. The four treatment-line biological samples are control-tolerant (CT), freezing-tolerant (FT), control-sensitive (CS), and freezing-sensitive (FS). Each compared combination is separated by an underscore (e.g., CT_FT). In the Venn diagram, the numbers of DEGs are illustrated across the intersection areas among the compared combinations. In total, we found 4550 DEGs from all the areas. Some of the areas are more important than others. Four critical areas, labeled I, II, III, and IV, totally contain 948 DEGs. Area I contains the tolerant treatment response (TTR) DEGs, excluding others. Area II contains the line response under freezing (LRF) DEGs, excluding others. Area III contains both tolerance treatment response and line response under freezing (TRLR) DEGs, excluding others. Area IV contains the treatment response (TR) DEGs within line.

Figure 4

Gene Ontology (GO) enrichment analysis of differentially expressed genes (DEGs). The GO analyses were performed on four sets of specific DEGs, labeled areas I, II, III, and IV in Figure 3; each area contained 99, 610, 70, and 169 DEGs, respectively. The observed gene frequency in each GO term is contrasted with the expected frequency under the null distribution (background/reference). The genes in areas II, I, III, and IV correspond to line response under freezing (LRF) (A), tolerance treatment response (TTR) (B), tolerance treatment response and line response under freezing (TRLR) (C), and freezing treatment response in both lines (TR) (D), respectively.

Figure 5

Validation of RNA-seq expression through qRT-PCR. Validation was performed in both freezing tolerant maize inbred line KR701 (A) and freezing sensitive maize inbred line Hei8834 (B). The plots demonstrate the expression ratio between before and after freezing treatment in Log scale with base of two.