DNA methylation regulates asymmetric zygote sub-genome activation in wheat

INTRODUCTION: Fertilization is an essential step in the life cycles of both animals and plants and gene transcription regulation during this process ensures its proper initiation. Chromatin remodeling, including DNA methylation, occurs in gametes and zygotes development as well as embryogenesis in diploid plants. However, how gene transcription is dynamically reprogrammed during fertilization in polyploid plants and whether DNA methylation is involved in orchestrating this process, remains poorly understood. OBJECTIVES: This study aimed to reveal the dynamic changes in zygotic genome activation (ZGA) during fertilization in hexaploid wheat and dissect the regulatory role of DNA methylation remodeling in the wheat ZGA process. METHODS: We systematically analyzed the transcriptome profiles (via RNA-seq) and genome-wide DNA methylation patterns (via whole-genome bisulfite sequencing, WGBS) of wheat gametes, 7 h-zygotes, and 24 h-zygotes aiming to reveal the interplay between DNA methylation and gene expression during fertilization. Semi-quantitative PCR was used to validate the accuracy of sample collection and the expression patterns of key genes, such as ECA1, E2F, and Wuschel. Additionally, CRISPR-Cas9-mediated genome editing was employed to verify whether the two homoeologs of VRN1 (a core vernalization gene) exhibit functional differentiation. RESULTS: We demonstrated that ZGA in hexaploid wheat is initiated prior to the first zygotic division. Notably, after fertilization, the proportion of genes with balanced expression among the A, B, and D subgenomes decreases significantly to 14.79%-27.92%, whereas the proportion of subgenome-dominant expression genes increases to 29.42%-51.13%, indicating extensive subgenome expression asymmetry during wheat ZGA. Concomitantly, we observed substantial remodeling of DNA methylation (including CG, CHG, and CHH contexts) with significant changes in proportion during the wheat ZGA process, confirming the involvement of epigenetic regulation. Intriguingly, the transcription of VRN1, a core gene controlling wheat vernalization and flowering time, was reset during fertilization, and this transcriptional reprogramming showed a strong correlation with dynamic changes in its DNA methylation levels. CONCLUSION: Our findings delineate the dynamic characteristics of ZGA in hexaploid wheat and reveal the critical role of DNA methylation remodeling in regulating ZGA and subgenome expression asymmetry during fertilization. The correlation between VRN1 transcriptional resetting and its DNA methylation status further highlights the specificity of epigenetic regulation in key functional genes. This study fills the gap in understanding epigenetic regulation of polyploid ZGA and provides novel epigenetic targets (e.g., VRN1 methylation sites) for improving wheat reproductive efficiency and breeding stable-yield varieties.