N6-methyladenosine RNA Modification Machinery: A Comparative Analysis Across Environmental Stress-Mediated and Developmental Barley Leaf Senescence Types

Senescence is a process that promotes phenotypic plasticity, enabling plants to adapt to changing environments. Developmental leaf senescence (DLS) represents an irreversible process, whereas dark-induced leaf senescence (DILS) is a reversible process. Our research aims to explain how epitranscriptomic machinery governs the irreversibility of senescence. N⁶-methyladenosine (m⁶A) is the most prevalent internal modification of messenger RNA. Its role in induced senescence adds a novel regulatory layer to the control of gene expression during this process. Here, we present the first genome-wide characterisation of m⁶A machinery regulatory genes — writers, readers, and erasers — in barley, and investigate their role in barley development and environmental stress response, using different leaf senescence types as a case study. We identified 31 m⁶A machinery-related genes and analyzed their phylogeny, domain organization, chromosomal distribution, and expression profiles across organs, developmental stages, and abiotic stresses. Transcriptomic profiling by RNA sequencing revealed distinct transcriptional responses of key regulators during DLS and DILS. In DILS, most writers and several readers were strongly upregulated, while erasers exhibited divergent expression patterns. Gene expression during DLS remained more stable, indicating limited m⁶A involvement. Quantification of global m⁶A RNA methylation confirmed increased methylation levels during DILS. Transcripts related to chloroplast function and stress response exhibited coordinated changes in mRNA methylation and expression. Together, these findings highlight a regulatory role of m⁶A RNA modification in premature leaf senescence and establish a framework for further exploration of RNA modifications in cereal crop improvement.