Harnessing R-loops biology for agriculture and food chemistry: From CUT&Tag to nanopore sequencing and beyond

ABSTRACT R-loops, three-stranded nucleic acid structures composed of an RNA: DNA hybrid and a displaced single-stranded DNA, have emerged as central regulators of transcription, chromatin organization, genome stability, and stress responses in plants. Over the past decade, R-loops detection technologies have undergone a paradigm shift from antibody-dependent approaches such as DRIP-seq to high-resolution, low-input methods including CUT&Tag, MapR, and R-ChIP, and culminating in nanopore sequencing, which enables direct, strand-specific, and long-read detection of RNA: DNA hybrids. This review provides the first comprehensive synthesis of R-loops mapping technologies specifically tailored to plant systems, with a distinctive focus on their translational applications in agriculture and food chemistry. We critically evaluate the strengths and limitations of each platform, tracing the technological trajectory from bulk, antibody-based profiling to single-cell-compatible, modification-aware, and real-time detection. We further propose that R-loops dynamics constitute a previously underappreciated nexus linking transcriptional regulation to food quality traits, including the biosynthesis of flavonoids, phenolic compounds, amino acids, and other bioactive metabolites that determine nutritional value, flavor, and post-harvest stability. By bridging plant molecular biology with food chemistry, this review demonstrates how R-loops mapping can inform precision breeding and CRISPR-based genome editing to enhance crop productivity, stress resilience, and food quality. We conclude by identifying critical gaps and outlining actionable future directions, positioning R-loops biology as an emerging pillar for sustainable agriculture and global food security.