Regulation of DNA (de)Methylation Positively Impacts Seed Germination during Seed Development under Heat Stress

Abstract

Seed development needs the coordination of multiple molecular mechanisms to promote correct tissue development, seed filling, and the acquisition of germination capacity, desiccation tolerance, longevity, and dormancy. Heat stress can negatively impact these processes and upon the increase of global mean temperatures, global food security is threatened. Here, we explored the impact of heat stress on seed physiology, morphology, gene expression, and methylation on three stages of seed development. Notably, Arabidopsis Col-0 plants under heat stress presented a decrease in germination capacity as well as a decrease in longevity. We observed that upon mild stress, gene expression and DNA methylation were moderately affected. Nevertheless, upon severe heat stress during seed development, gene expression was intensively modified, promoting heat stress response mechanisms including the activation of the ABA pathway. By analyzing candidate epigenetic markers using the mutants' physiological assays, we observed that the lack of DNA demethylation by the ROS1 gene impaired seed germination by affecting germination-related gene expression. On the other hand, we also observed that upon severe stress, a large proportion of differentially methylated regions (DMRs) were located in the promoters and gene sequences of germination-related genes. To conclude, our results indicate that DNA (de)methylation could be a key regulatory process to ensure proper seed germination of seeds produced under heat stress.

Keywords: DNA methylation; heat stress; seed; seed development; seed germination.

Figure 1

Physiological characteristics of A. thaliana seeds under heat stress development. (A) Mature seed water content measured every two days for a duration of 14 days. Two-way ANOVA showing the difference between stress conditions and control conditions at each time point, therefore 25 °C and 27 °C stress treatment against 21 °C. (B) Protein content based on nitrogen content. Two-way ANOVA comparing 23 °C, 25 °C, and 27 °C against 21 °C. (C) Percentage of fresh seed germination in which the numbers inside bars represent the decrease in seed germination upon heat stress conditions compared to control temperatures. Two-way ANOVA comparing 23 °C, 25 °C, and 27 °C against 21 °C. (D) Seed longevity based on P50, in which artificial aging was imposed for 6, 10, 14, 21, and 28 days. (E) Exemplary electronic microscopy of Col-0 seeds grown at 23 °C, 25 °C, and 27 °C. The below panel of the 27 °C condition shows a wrinkled-shape seed. Asterisks represent p-value significance: * p < 0.05; ** p < 0.001; *** p < 0.0001. Standard deviation is shown in (A–C) graphs.

Figure 2

Transcriptomics analysis of Col-0 seeds developed under heat stress conditions. (A) RNA-Seq samples were obtained during embryo developmental stages showed here in time-course order: Heart (H), Bent (B), and Mature (M). Average temperature of 23 °C was used as control against Mild Stress (MS) at 25 °C and against Severe Stress (SS) at 27 °C. (B) Scatter plot showing the quantity of differentially expressed genes (DEGs) for MS in orange and SS in red. (C) Venn diagrams of DEGs from SS at each embryo developmental stage. Upper panels show upregulated genes and lower panels show downregulated genes.

Figure 3

Gene set enrichment (GSEA) analysis of DEGs in Col-0 seeds under severe heat stress. (A) Functional enriched GO terms from upregulated differentially expressed genes. (B) Functional enriched GO terms from downregulated DEGs. The size of the dot represents the gene count. The totality of differentially up- or downregulated genes in each stage was used to perform a hypergeometric test, and the p-values were converted to false discovery rate (FDR)-corrected p-value as shown in colors, the red color being more significant than the blue color. H = heart, B = bent, M = mature, and C = common to all stages.

Figure 4

Evaluation of CMT3 and ROS1 expression and physiology of cmt3 and ros1-4 mutants. (A) Gene expression in TPM of CMT3 and ROS1 at heart, bent, and mature stages of embryo development during control temperature (23 °C), MS (25 °C) and SS (27 °C). CMT3 is represented by white bars while ROS1 is represented by grey bars. Standard deviation is shown. Two-way ANOVA comparing differences in gene expression at 23 °C, 25 °C, and 27 °C for each genotype at the same stage. Letters represent statistically significant differences with a p-value < 0.001. Letters a or b used for ROS1 and letters a’ or b’ used for CMT3. (B) Seed germination for wild-type (Col-0 or WS) and mutant (ros1-4 or cmt3) genotypes at 23 °C (green) and 27 °C (red) average temperature. (C) Panels show embryos and seed coats from wild-types (Col-0 or WS) and mutant (ros1-4 or cmt3) genotypes at 23 °C and 27 °C average temperature. The upper panel is composed of electronic microscopy photos for phenotype observations and the lower panel shows embryo survival evaluation with TTZ. ros1-4 at 27 °C was divided into normal-shaped seeds (N) or wrinkled-shaped seeds (W). (D) Seed measurements of length and width in micrometers. Boxes and whiskers showing minimum and maximum with median line from all observations. Green bars represent temperature at 23 °C and red bars represent the temperature at 27 °C (SS). Two-way ANOVA comparing difference size between wild-types (Col-0 or WS) and mutants (ros1-4 or cmt3), respectively, at 23 °C wild-type against 23 °C mutant, and 27 °C wild-type against 27 °C mutant, therefore green against green and red against red. Letters represent statistically significant differences with a p-value < 0.001. Letters a or b used for length measures and letters a’ or b’ used for width measures. Percentage of wrinkle-shaped seeds for each genotype at 23 °C and 27 °C is shown at the base of the x-axis.

Figure 5

Methylome analysis and DMRs related to germination genes. (A) Percentage of cytosine methylation per context (CG, CHG, CHH) on bent and mature embryo stages of Col-0 seeds grown under the Control temperature (C = 23 °C) or Mild Stress (MS = 25 °C), or Severe Stress (SS = 27 °C). (B) Amount of DMRs between control condition and MS or control condition and SS in all contexts (CH, CHG, CHH) for bent and mature stages at 1 Kb promoter regions (P) and gene sequences (G). (C) Normalized expression (TPM) heat-map of 39 genes differentially expressed during seed germination and containing a DMR at mature stage with severe heat stress (MethDEGs). Expression in Col-0 seeds at conditions: fresh seed (FS); dried seed (DS); stratified and imbibed seeds at 4 °C (St) for 1 h, 12 h and 48 h; stratified and imbibed seeds at 20 °C light for germination (Ger) for 1 h, 6 h, 12 h, 24 h, and 48 h. (D) Differential expression analysis of MethDEGs between ros1-4 and Col-0, comparing dry seeds against two germination stages (II and IV), whereas green indicates upregulated genes and red indicates downregulated genes in ros1-4.