Integrative mapping of chloroplast rRNA modifications reveals conserved and lineage-specific features and stepwise modification during plastid ribosome biogenesis

Abstract Background Ribosomal RNA (rRNA) modifications contribute to ribosome structure and function, yet their distribution and roles in chloroplast ribosomes remain poorly defined. Previous studies have focused on individual enzymes or sites, leaving the global modification landscape and its evolutionary context largely unresolved. Results Here we present an integrative analysis of post-transcriptional modifications in chloroplast 16 S and 23 S rRNAs of Arabidopsis thaliana . By combining direct RNA sequencing with complementary cDNA-based approaches and meta-analysis of publicly available RNA-seq datasets, we identified seven modified sites in 16 S rRNA and ten in 23 S rRNA with high confidence. Most modifications correspond to conserved bacterial counterparts, indicating that plastid ribosomes retain a prokaryotic-type functional core. In addition, we provide evidence for a methylated cytosine at position C1940 in 23 S rRNA that appears to be conserved within the plant lineage but absent from bacteria, suggesting plastid-specific adaptation. Functional analysis of PFC1, the plastid orthologue of the bacterial dimethyltransferase KsgA, showed that loss of N ⁶, N ⁶-dimethyladenosine formation impairs 16 S rRNA maturation, delays chloroplast ribosome assembly and affects photosynthetic performance in young leaves. Single-molecule DRS analysis of defined 16 S and 23 S rRNA processing states further revealed stage-specific accumulation of modification-associated signatures, consistent with stepwise installation of chloroplast rRNA modifications during ribosome biogenesis. Conclusions Our results provide a comprehensive and experimentally supported map of chloroplast rRNA modifications and show that plastid rRNA modification is linked to rRNA maturation and ribosome assembly. This work establishes a framework for investigating how conserved and lineage-specific rRNA modifications contribute to plastid ribosome function, chloroplast development and plant responses to environmental conditions.