Enhancers associated with unstable RNAs are rare in plants

Unstable transcripts have emerged as markers of active enhancers in vertebrates and shown to be involved in many cellular processes and medical disorders. However, their prevalence and role in plants is largely unexplored. Here, we comprehensively captured all actively initiating ("nascent") transcripts across diverse crops and other plants using capped small (cs)RNA-seq. We discovered that unstable transcripts are rare, unlike in vertebrates, and often originate from promoters. Additionally, many "distal" elements in plants initiate tissue-specific stable transcripts and are likely bone fide promoters of yet-unannotated genes or non-coding RNAs, cautioning against using genome annotations to infer "enhancers" or transcript stability. To investigate enhancer function, we integrated STARR-seq data. We found that annotated promoters, and other regions that initiate stable transcripts rather than unstable transcripts, function as stronger enhancers in plants. Our findings underscore the blurred line between promoters and enhancers and suggest that cis-regulatory elements encompass diverse structures and mechanisms in eukaryotes.