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.

The biological implications of R-loops are increasingly receiving attention in plant biology. Compared with recent progresses in humans, the studies of R-loops in plants are lagging behind. Massive efforts must be invested by the plant community to better harness the possible regulatory functions of R-loops. Pioneering studies indicate that R-loops might act as key regulators of plant growth and development, and stress responses. This review thus timely updates the biological implications of R-loops and proposes a series of future lines of investigations to further decipher R-loop biology in plants. The following four aspects are included: we discuss here about the interplay between R-loops and epigenetic modifications at DNA, RNA and chromatin levels; about their regulatory roles in genome integrity, centromere functions and DNA replication; and about how they likely control plant growth and development as well as stress responses and RNA processing. Altogether, the wealth of information provided here portrays R-loop biology in plants accurately, and makes these new regulators interesting genetic levers to actuate in the aim of providing plants with new and beneficial agronomical traits.