Ribosome profiling reveals distinct translational programs underlying Arabidopsis seed dormancy and germination

Abstract

Seed dormancy and germination represent a critical developmental transition that determines plant fitness, yet the contribution of translational regulation to this process remains poorly understood. Here, we used genome-wide ribosome profiling (Ribo-seq) combined with RNA sequencing (RNA-seq) to investigate how translational control shapes the transition from dormancy to germination in Arabidopsis thaliana seeds. We analyzed dry dormant seeds, stratified non-dormant seeds, and seeds during early imbibition, enabling simultaneous assessment of transcript abundance and ribosome occupancy. Our analyses reveal that dry seeds harbor an unexpectedly organized translational machinery, with ribosomes pre-positioned at start codons and within coding regions of thousands of stored mRNAs, indicating a poised translational state. Dormancy release and early imbibition triggered extensive gene-specific changes in translational efficiency that were largely uncoupled from transcript abundance, highlighting selective translation as a key regulatory layer. Genes involved in ribosome biogenesis, protein folding, and hormone signaling were preferentially translated during dormancy maintenance, whereas germination-promoting factors showed increased ribosome occupancy following stratification. Global ribosome profiling further uncovered dynamic ribosome pausing at stop codons and pronounced modulation of translation initiation during imbibition.We also identified widespread translation of upstream open reading frames (uORFs) and demonstrated that uORF-mediated repression constitutes a major translational checkpoint during seed imbibition. Functional assays confirmed that uORFs from MARD1 and PAO4 repress downstream translation in vivo. Together, our results establish translational regulation as a central mechanism governing seed dormancy and germination, revealing how ribosome positioning and uORF activity fine-tune protein synthesis to control developmental transitions in response to environmental cues.