Comparative and functional analysis unveils the contribution of photoperiod to DNA methylation, sRNA accumulation, and gene expression variations in short‐day and long‐day grasses.

SUMMARY: Photoperiod employs complicated networks to regulate various developmental processes in plants, including flowering transition. However, the specific mechanisms by which photoperiod affects epigenetic modifications and gene expression variations in plants remain elusive. In this study, we conducted a comprehensive analysis of DNA methylation, small RNA (sRNA) accumulation, and gene expressions under different daylengths in facultative long‐day (LD) grass Brachypodium distachyon and short‐day (SD) grass rice. Our results showed that while overall DNA methylation levels were minimally affected by different photoperiods, CHH methylation levels were repressed under their favorable light conditions, particularly in rice. We identified numerous differentially methylated regions (DMRs) that were influenced by photoperiod in both plant species. Apart from differential sRNA clusters, we observed alterations in the expression of key components of the RNA‐directed DNA methylation pathway, DNA methyltransferases, and demethylases, which may contribute to the identified photoperiod‐influenced CHH DMRs. Furthermore, we identified many differentially expressed genes in response to different daylengths, some of which were associated with the DMRs. Notably, we discovered a photoperiod‐responsive gene MYB11 in the transcriptome of B. distachyon, and further demonstrated its role as a flowering inhibitor by repressing FT1 transcription. Together, our comparative and functional analysis sheds light on the effects of daylength on DNA methylation, sRNA accumulation, and gene expression variations in LD and SD plants, thereby facilitating better designing breeding programs aimed at developing high‐yield crops that can adapt to local growing seasons. Significance Statement: Our comprehensive analysis of DNA methylation, small RNA accumulation, and gene expressions combined with functional analysis of long‐day (LD) photoperiod‐induced MYB11 in Brachypodium distachyon offers insights into the impact of daylength on epigenetic modifications, and gene expression variations in LD and short‐day plants. Our research fosters the potential for more calculated scheming of breeding programs that strive to produce high‐yielding crops capable of adapting to their respective local growing seasons. [ABSTRACT FROM AUTHOR]