Nitrogen signaling genes and SOC1 determine the flowering time in a reciprocal negative feedback loop in Chinese cabbage (Brassica rapa L.) based on CRISPR/Cas9-mediated mutagenesis of multiple BrSOC1 homologs

Abstract

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 (Brassica rapa 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, nitrite reductase (BrNIR) and nitrate reductase (BrNIA), 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 Circadian 1 (BrCIR1) was significantly up-regulated in line “50”, whereas the flowering enhancer genes named SUPPRESSOR OF OVEREXPRESSION OF CONSTANCE 1 homologs (BrSOC1s) were substantially up-regulated in line “4004”. CRISPR/Cas9-mediated mutagenesis in Chinese cabbage demonstrated that the BrSOC1-1/1-2/1-3 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 (BrNIA1 and BrNIR1) and BrSOC1s to control flowering time and positive feedback control of the expression of BrSOC1s.