A sensitive synthetic reporter for visualizing cytokinin signaling output in rice

Cytokinins play many essential roles in plant growth and development, mainly through signal transduction pathways. Although the cytokinin signaling pathway in rice has been clarified, no synthetic reporter for cytokinin signaling output has been reported for rice. The sensitive synthetic reporter two-component signaling sensor (TCSn) is used in the model plant Arabidopsis; however, whether the reporter reflects the cytokinin signaling output pattern in rice remains unclear. Early-cytokinin-responsive type-A OsRR-binding element (A/G)GAT(C/T) was more clustered in the 15 type-A OsRRs than in the 13 control genes. Quantitative polymerase chain reaction analysis showed that the relative expression of seven type-A OsRRs in roots and shoots was significantly induced by exogenous cytokinin application, and that of seven OsRRs, mainly in roots, was inhibited by exogenous auxin application. We constructed a transgenic rice plant harboring a beta-glucuronidase (GUS) driven by the synthetic promoter TCSn. TCSn::GUS was expressed in the meristem of germinated rice seed and rice seedlings. Furthermore, TCSn::GUS expression in rice seedlings was induced specifically by exogenous cytokinin application and decreased by exogenous auxin application. Moreover, no obvious reduction in GUS levels was observed after three generations of selfing of transgenic plants, indicating that TCSn::GUS is not subject to transgene silencing. We report here a robust and sensitive synthetic sensor for monitoring the transcriptional output of the cytokinin signaling network in rice.