An R‐loop is a three‐stranded chromatin structure that consists of a displaced single strand of DNA and an RNA:DNA hybrid duplex, which was thought to be a rare by‐product of transcription. However, recent genome‐wide data have shown that R‐loops are widespread and pervasive in a variety of genomes, and a growing body of experimental evidence indicates that R‐loops have both beneficial and harmful effects on an organism. To maximize benefit and avoid harm, organisms have evolved several means by which they tightly regulate R‐loop levels. Here, we summarize our current understanding of the biogenesis and effects of R‐loops, the mechanisms that regulate them, and methods of R‐loop profiling, reviewing recent research advances on R‐loops in plants. Furthermore, we provide perspectives on future research directions for R‐loop biology in plants, which might lead to a more comprehensive understanding of R‐loop functions in plant genome regulation and contribute to future agricultural improvements.
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