Nitrogen Signaling Genes and Determine the Flowering Time in a Reciprocal Negative Feedback Loop in Chinese Cabbage ( L.) Based on CRISPR/Cas9-Mediated Mutagenesis of Multiple Homologs.

Precise flowering timing is critical for the plant life cycle. Here, we examined the molecular mechanisms and regulatory network associated with flowering in Chinese cabbage ( L.) by comparative transcriptome profiling of two Chinese cabbage inbred lines, "4004" (early bolting) and "50" (late bolting). RNA-Seq and quantitative reverse transcription PCR (qPCR) analyses showed that two positive nitric oxide (NO) signaling regulator genes, () and (), were up-regulated in line "50" with or without vernalization. In agreement with the transcription analysis, the shoots in line "50" had substantially higher nitrogen levels than those in "4004". Upon vernalization, the flowering repressor gene () was significantly up-regulated in line "50", whereas the flowering enhancer genes named homologs () were substantially up-regulated in line "4004". CRISPR/Cas9-mediated mutagenesis in Chinese cabbage demonstrated that the genes were involved in late flowering, and their expression was mutually exclusive with that of the nitrogen signaling genes. Thus, we identified two flowering mechanisms in Chinese cabbage: a reciprocal negative feedback loop between nitrogen signaling genes ( and ) and to control flowering time and positive feedback control of the expression of .