Abstract
The dynamic organization of chromatin governs gene expression by regulating DNA accessibility. In plants, light not only initiates photomorphogenesis but also reshapes higher-order chromatin architecture. However, the limited resolution of current techniques impedes investigation of chromatin dynamics at the single-molecule level. Here, we applied Fiber-seq, a long-read, single-molecule chromatin profiling method, to construct near-nucleotide resolution maps of chromatin accessibility, nucleosome positioning, and cytosine methylation in Arabidopsis thaliana and maize. We observed that light exposure during photomorphogenesis led to significant, locus-specific changes in chromatin accessibility-both increases and decreases-especially in genes related to photosynthesis, hormone signaling, and development. Analysis of chromatin accessibility changes in cop1-6, pifq, and hy5 hyh mutants revealed that classical light signaling pathways regulate chromatin accessibility. Additionally, using high-fidelity long-read sequencing, we profiled DNA methylation in previously inaccessible repetitive regions such as 5S rRNA gene clusters and CEN180 satellite repeats. These heterochromatic loci exhibited distinct light-dependent changes in chromatin accessibility that were undetectable using prior methods. In maize, we demonstrated that Fiber-seq identifies a broader range of biologically relevant open chromatin regions, enabling both high-accuracy de novo genome assembly and the detection of fine-scale structural variants. Collectively, Fiber-seq offers an integrated view of chromatin states across regulatory and repetitive elements, providing critical insights into how environmental signals reshape plant epigenomes.