Position and Strand Identity of G-Quadruplex Motifs Are Associated with Distinct Functional Signatures in Plants

Putative G-quadruplex (G4) sequences have been linked to transcriptional regulation in individual plant species, but a systematic, comparative view across the plant kingdom has been lacking. In this study, we analyzed putative G4-forming sequences (PQS) near transcription start sites (TSSs) in 70 plant species spanning over 1.2 billion years of evolutionary divergence, from algae to angiosperms. Using the G4Hunter algorithm, putative G4 motifs were mapped and scored separately on the coding and template DNA strands. This strand-resolved analysis identified four position/strand categories, among which template-strand motifs downstream of the TSS were the most abundant and resistant to an increase in the G4Hunter threshold. The majority of PQS-positive genes (62.4%) carry a motif in only one category, although combinatorial patterns involving two or more categories also occur. Gene Ontology enrichment analysis of Arabidopsis thaliana homologs tested in each species revealed two main recurrent patterns of modest effect size: downstream template-strand motifs were associated with chloroplast and mRNA-binding terms, and upstream template-strand motifs were associated with transcription factor activity and regulation of transcription, whereas downstream coding-strand motifs showed a weaker signal. At the level of individual genes, position-specific G4s recurred among homologs more often than expected at random, mostly downstream, where this may partly reflect the conservation of the coding sequence. These associations, based on computational prediction, provide a starting point for prioritizing candidate G4 elements for experimental validation of their role in transcriptional control in plants.