Supplemental Material for Yang et al., 2020

Circular RNA (circRNA) is a covalently-closed single-stranded RNA molecule that plays an important role in transcriptional regulation of gene expression in a variety of species. Light intensity is a pivotal environmental factor affecting plant growth and development. However, little is known regarding photoresponsive plant circRNAs. Here, we aimed to investigate the expression and function of circRNAs in lettuce leaves in response to different light intensity treatments. We performed RNA sequencing (RNA-Seq) on leaves of lettuce (Lactuca sativa) to determine circRNA expression profiles and reverse-transcription polymerase chain reaction (PCR) to validate the candidate circRNA molecules. We then combined bioinformatics approach to explore the function of the parental genes of circRNA, including network, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes pathway analysis. We identified 1650 circRNAs in lettuce, of which 1508 (86.40%) were derived from exons. Using real-time PCR, we characterized 10 validated differentially expressed circRNAs and their parental genes, all of which showed expression patterns consistent with RNA-Seq data. Interestingly, the expression of circRNA was, in some cases, inversely correlated with the expression of the parental gene. Furthermore, analysis of the circRNA–microRNA–mRNA network suggests that circRNAs may be involved in plant hormone signaling and chlorophyll metabolism during photoreactivity. These findings provide an essential reference basis for studying circRNAs’ biological mechanisms in light-treated plants.