Comprehensive Profiling of the miRNome and Degradome Reveals Regulatory Signatures of Seed Aging and Germination

Small noncoding RNAs are recognized as crucial regulators of seed germination, but their role in seed aging remains unclear. To address this, we performed RNA sequencing (RNA-seq) on barley (Hordeum vulgare L.) seeds with varying viability levels after long-term storage in hermetically sealed containers since the 1972 harvest. This globally unique material, characterized by genetic homogeneity and contrasting germination capacities, enabled an in-depth analysis of microtranscriptomic changes during germination. We identified 62 known miRNAs from 11 families and 234 novel miRNAs, with miR159, miR168, and miR166 showing consistently high expression across all germination stages and viability groups. Differential expression analysis revealed 28 miRNAs whose abundance varied significantly with seed viability and germination phase. Functional predictions supported by quantitative reverse transcription PCR (qRT-PCR) and degradome-based target identification indicated that these miRNAs regulate key developmental and metabolic pathways. Several isomiRs exhibited sample-specific expression, suggesting the viability-dependent activation of distinct molecular mechanisms. Gene Ontology analysis highlighted processes related to nucleic acid binding, nuclear organization, and cytoplasmic metabolism as central during germination. We propose that miRNA profiles may reflect an "epigenetic inheritance"-a molecular memory of aging stored in seeds-rather than solely a response to current conditions. This concept may help explain aging-related phenotypes such as delayed germination and reduced vigor, warranting further investigation.