CmNLP7 interacts with CmPP6 to suppress flowering time in Chrysanthemum

Nitrogen (N) is a key component in plants and their biological macromolecules, having a profound effect on developmental stages, such as germination, vegetative growth, and flowering. However, the mechanism of nitrogen-regulated flowering time remains unclear. In this study, CmNLP7 was isolated from the chrysanthemum cultivar ‘Jinba’ and was characterized. CmNLP7 is a transcription factor localized in the nucleus but has no transcriptional activity. Tissue expression pattern analysis showed that CmNLP7 was mainly transcribed in leaves and roots. Knocking down CmNLP7 through the artificial-miRNA method in chrysanthemum resulted in early flowering under optimal nitrogen (ON) and low nitrogen (LN) conditions; whereas overexpression lines showed delayed flowering under LN conditions. Transcriptome sequencing analysis showed that the nitrate transporters NRT2.5, NPF3.1, and NPF4.6; SBP-like genes SPL7 and SPL12, and flowering integration factor FT were significantly up-regulated in the knockdown lines. Based on the KEGG pathway enrichment analysis, the differentially transcribed genes were enriched in phenylpropanoid biosynthesis and starch and sucrose metabolism pathways, which indicated their alleged function in nitrogen-regulated flowering and development in chrysanthemum. Furthermore CmPP6 as a homolog of the Arabidopsis phosphatase PP6, was verified as an interacting protein of CmNLP7 by yeast two-hybrid, BiFC, pull-down and Biacore in vitro and in vivo, and the knockdown line of CmPP6 (amiR-CmPP6) flowered earlier compared to that of the wild-type chrysanthemum ‘Jinba’. Collectively, these results demonstrated that CmPP6 interacts with CmNLP7 to regulate chrysanthemum flowering, and CmNLP7 could regulate flowering time in response to nitrogen, which lays a foundation for the regulation of flowering and molecular breeding of chrysanthemum through changes in nutrient signaling.