Minimal evidence for consistent changes in maize DNA methylation patterns following environmental stress

Abstract

DNA methylation is a chromatin modification that is sometimes associated with epigenetic regulation of gene expression. As DNA methylation can be reversible at some loci, it is possible that methylation patterns may change within an organism that is subjected to environmental stress. In order to assess the effects of abiotic stress on DNA methylation patterns in maize (Zea mays), seeding plants were subjected to heat, cold, and UV stress treatments. Tissue was later collected from individual adult plants that had been subjected to stress or control treatments and used to perform DNA methylation profiling to determine whether there were consistent changes in DNA methylation triggered by specific stress treatments. DNA methylation profiling was performed by immunoprecipitation of methylated DNA followed by microarray hybridization to allow for quantitative estimates of DNA methylation abundance throughout the low-copy portion of the maize genome. By comparing the DNA methylation profiles of each individual plant to the average of the control plants it was possible to identify regions of the genome with variable DNA methylation. However, we did not find evidence of consistent DNA methylation changes resulting from the stress treatments used in this study. Instead, the data suggest that there is a low-rate of stochastic variation that is present in both control and stressed plants.

Keywords: DNA methylation; abiotic stress; epigenetics; maize; tissue culture.

Figure 1

Experimental design flow-chart. (A) B73 seedlings (all derived from a single self-pollination) were grown for 14 days. Some of these plants were then subjected to cold, heat, or UV-B stress for 4 h per day (every other day for four treatments). At the conclusion of the treatment regime the stressed plants were then grown under standard conditions to maturity. DNA was isolated from the flag leaf (the last adult leaf initiated before the tassel) and used to perform meDIP-chip. (B) Tissue culture samples from the genotype A188 was used to perform regeneration. The plantlets were then grown to maturity (along with sibling non-tissue culture A188 plants) and tissue was collected from the flag leaf for meDIP-chip.

Figure 2

Analysis of DNA methylation gains and losses in DMRs. Three pie-charts (proportionally sized based on the number of DMRs for each chart) are used to show the frequency of hypermethylation (blue), hypomethylation (yellow), or both (red) relative to the control average value. The top pie-chart shows DMRs that are only present in stressed samples, the middle chart shows DMRs that are only found in control individuals and the bottom pie-chart shows DMRs that are found in both stress and control plants.

Figure 3

Location of DMRs relative to genes and TEs. Each DMR was classified based on whether it exhibits hypomethylation, hypermethylation, or both hypo- and hyper-methylation relative to the control average (x-axis). The DMRs were also classified based on whether they exhibit significant differences in stress samples only, control samples only, or both control and stress (y-axis). Proportionally sized pie-charts are used to illustrate the frequencies of DMRs located near (<500 bp) genes (blue), TEs (black), both genes and TEs (red), or neither (yellow).

Figure 4

Clustering of DNA methylation differences in plants subjected to abiotic stress. Hierarchical clustering (Wards method) was performed for the DMRs identified in stressed plants (A), control plants (B), or both stress and control plants (C). The heatmap uses red to indicate DNA methylation levels higher than the control average and blue to indicate lower DNA methylation levels. The samples are indicated at the bottom of each plot and the symbols indicate the treatment (green-control, blue-cold, red-heat, yellow-UV).

Figure 5

Clustering of DNA methylation differences in plants subjected to abiotic stress or tissue culture. Hierarchical clustering (Wards method) was performed for the DMRs identified the analysis of individual plants subjected to abiotic stress or tissue culture. These DMRs are the subset that do not exhibit differences in B73 (used for abiotic stresses) relative to A188 (used for tissue culture). The heatmap uses red to indicate DNA methylation levels higher than the control average and blue to indicate lower DNA methylation levels. The samples are indicated at the bottom of each plot and the symbols indicate the treatment (green squares, B73 control; orange squares, A188 control; blue triangles, cold; red circles, heat; yellow stars, UV; orange pluses, tissue culture regenerants).