DNA G-quadruplexes (G4s) are non-canonical four-stranded DNA structures involved in various biological processes in eukaryotes. Molecularly crowded solutions and monovalent cations have been reported to stabilize in vitro and in vivo G4 formation. However, how K
+ and Na
+ affect G4 formation genome-wide is still unclear in plants. Here, we conducted BG4-DNA-IP-seq, DNA immunoprecipitation with anti-BG4 antibody coupled with sequencing, under K
+ and Na
+ + PEG conditions in vitro. We found that K
+-specific IP-G4s had a longer peak size, more GC and PQS content, and distinct AT and GC skews compared to Na
+-specific IP-G4s. Moreover, K
+- and Na
+-specific IP-G4s exhibited differential subgenomic enrichment and distinct putative functional motifs for the binding of certain trans-factors. More importantly, we found that K
+-specific IP-G4s were more associated with active marks, such as active histone marks, and low DNA methylation levels, as compared to Na
+-specific IP-G4s; thus, K
+-specific IP-G4s in combination with active chromatin features facilitate the expression of overlapping genes. In addition, K
+- and Na
+-specific IP-G4 overlapping genes exhibited differential GO (gene ontology) terms, suggesting they may have distinct biological relevance in rice. Thus, our study, for the first time, explores the effects of K
+ and Na
+ on global G4 formation in vitro, thereby providing valuable resources for functional G4 studies in rice. It will provide certain G4 loci for the biotechnological engineering of rice in the future.
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