The presence and impact of G-quadruplexes in plant chloroplast DNA

Chloroplasts are essential organelles in the regulation of oxygen metabolism in algae and plants, and therefore play a central role in the physiology of oxygen‑dependent life. Chloroplasts typically contain a circular DNA molecule with replication and inheritance largely independent of the nuclear genome. Putative G‑quadruplex–forming sequences (PQS) have emerged as important non‑canonical DNA features in various genomes; however, their distribution and potential roles in chloroplast DNA (cpDNA) remain poorly understood. In this study, we used the G4Hunter algorithm to perform a comprehensive, large‑scale analysis of PQS across all publicly available chloroplast genomes in the NCBI database (n = 7,187). Our analysis provides a systematic characterization of cpDNA with respect to PQS localisation, frequency, conservation, and variation across major evolutionary lineages. The average frequency of PQS in cpDNA genomes was 0.873 PQS per kilobase pair (kbp), ranging from 0.018 PQS/kbp in Sonderella linearis to 5.011 PQS/kbp in Paradoxia multiseta. Marked lineage-specific differences were observed, with the highest PQS frequencies in lycophytes and the lowest in red algae. PQS were unevenly distributed along cpDNA sequences, with enrichment depending on motif length and genomic context. Specifically, PQS were preferentially located within transposable elements, replication origins, tRNA genes, 3′ untranslated regions (3′UTRs), and repeat regions, rather than being randomly dispersed. Our findings reveal non-random patterns of PQS distribution across chloroplast genomes and identify genomic regions with consistent enrichment of these motifs. While these observations are consistent with the possibility that PQS could contribute to structural or regulatory processes in cpDNA, their functional relevance remains to be experimentally validated. This work provides a comprehensive resource and establishes a foundation for future studies aimed at elucidating the biological roles of G‑quadruplex structures in chloroplast genomes.