CircZmMED16 delays plant flowering by negatively regulating starch content through its binding to ZmAPS1

Circular RNAs (circRNAs), a type of head-to-tail closed RNA molecules, have been implicated in various aspects of plant development and stress responses through transcriptome sequencing; however, the precise functional roles of circRNAs in plants remain poorly understood. In this study, we identified a highly expressed circular RNA, circZmMED16, derived from exon 8 of the mediator complex subunit 16 (ZmMED16) across different maize (Zea mays L.) inbred lines using circRNA-seq analysis. This circRNA is predominantly expressed in maize tassels and functions in the cytoplasm. Overexpression of circZmMED16 resulted in increased expression of ZmMED16/AtMED16 and delayed flowering in both maize and Arabidopsis thaliana, compared with that in wild-type plants. In contrast, overexpression of the parent gene ZmMED16 did not alter the flowering time of transgenic plants in Arabidopsis, suggesting that circZmMED16 plays a specific role in regulating flowering, distinct from that of linear ZmMED16. To further understand the mechanisms underlying the regulation of flowering time by circZmMED16, we performed RNA pull-down, dual-luciferase, RNA interference (RNAi), and ribonuclease protection assays (RPA). These results indicate that circZmMED16 interacts with small subunit 1 of ADP-glucose pyrophosphorylase (APS1) mRNA in both maize and Arabidopsis. The knockdown of circZmMED16 increased the expression of ZmAPS1, whereas the overexpression of circZmMED16 led to the downregulation of ZmAPS1 RNA and protein. By affecting ZmAPS1 expression, circZmMED16 reduced ADP-glucose pyrophosphorylase (AGPase) activity and led to delayed flowering. These results revealed a novel regulatory mechanism for circRNAs in flowering time and shed light on their functional and regulatory roles in plants.