RNA i-motif landscapes in plant kingdom and their potential functional roles.

Nucleotide composition has evolved in patterns that reflect both phylogenetic divergence and environmental adaptation. In plants, our previous analysis of transcriptomes from the 1,000 Plants (1KP) initiative revealed that nucleotide frequencies are significantly associated with habitat temperatures, suggesting selective pressures on RNA sequence and structure. Such pressures may promote the emergence of specific non-canonical RNA motifs, exemplified by G-rich RNA G-quadruplexes, which have been implicated in plant environmental adaptation. Whether similar evolutionary selection operates on other nucleotides and RNA structural motifs, however, remains unclear. Here, we investigate the evolutionary landscape of the RNA i-motif (iM), a C-rich non-canonical RNA structure, across the plant kingdom. Using iM-Seeker, we systematically identified RNA iMs and uncovered a pronounced enrichment within 5' untranslated regions (5'UTRs), with monocots exhibiting the highest iM abundance among major plant lineages. Integration of ecological variables with iM densities revealed that species from warmer environments preferentially harbor increased numbers of 5'UTR iMs, a trend most evident in monocots. Our translatome analyses in rice, wheat, tomato, and maize further indicate that 5'UTR iMs are generally associated with translational repression. Consistently, experimental validation using orthologous monocot 5'UTRs confirmed that RNA iMs are capable of repressing translation. Together, these findings reveal evolutionary selection on nucleotide composition and RNA structural motifs, highlight the adaptive significance of RNA i-motifs, and suggest that plants may use i-motifs as molecular signatures to facilitate environmental adaptation during evolution.