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.

Basic helix–loop–helix (bHLH) transcription factor gene family in plants controls various growth and development aspects; however, the actual roles of these genes in flowering plants are not well known. In this study, a novel bHLH protein CmbHLH110 was found to interact with CmERF110 by in vitro and in vivo experiments, a chrysanthemum ERF110 homolog that acts as a positive flowering regulator. In addition, CmbHLH110 was also found to regulate the flowering of chrysanthemums, overexpression of CmbHLH110 causes chrysanthemums to flower earlier, and suppressed CmbHLH110 leads to delayed flowering. Furthermore, the loss-of-function Arabidopsis mutant of its homologue PERICYCLE FACTOR TYPE-A 5 (PFA5) had a noticeable late flowering phenotype, and CmbHLH110 completely complemented the late flowering phenotype of the pfa5 mutant, whereas heterologous overexpression of CmbHLH110 in Arabidopsis Col-0 caused early flowering. Transcriptome sequencing revealed significant differential expression of flowering-related and circadian clock-related genes in transgenic chrysanthemum. Therefore, we concluded that CmbHLH110, as a novel flowering regulator, could interact with CmERF110 to regulate flowering in chrysanthemum.

The quantitative control of FLOWERING LOCUS T (FT) activation is important for the floral transition in flowering plants. However, the flowering regulation mechanisms in the day-neutral, summer-flowering chrysanthemum plant remain unclear. In this study, the chrysanthemum BBX7 homolog CmBBX7 was isolated and its flowering function was identified. The expression of CmBBX7 showed a diurnal rhythm and CmBBX7 exhibited higher expression levels than CmBBX8. Overexpression of CmBBX7 in transgenic chrysanthemum accelerated flowering, whereas lines transfected with a chimeric repressor (pSRDX-CmBBX7) exhibited delayed flowering. Yeast single hybridization, luciferase, electrophoretic mobility shift, and chromatin immunoprecipitation assays showed that CmBBX7 directly targets CmFTL1. In addition, we found that CmBBX7 and CmBBX8 interact to positively regulate the expression of CmFTL1 through binding to its promoter. Collectively, these results highlight CmBBX7 as a key cooperator in the BBX8–FT module to control chrysanthemum flowering.

Flowering is an important process in higher plants and is regulated by a variety of factors, including light, temperature, and phytohormones. Flowering restriction has a considerable impact on the commodity value and production cost of many horticultural crops. In Arabidopsis, the FT/TFL1 gene family has been shown to integrate signals from various flowering pathways and to play a key role in the transition from flower production to seed development. Studies in several plant species of the FT/TFL1 gene family have revealed it harbors functional diversity in the regulation of flowering. Here, we review the functional evolution of the FT/TFL1 gene family in horticulture plants and its unique regulatory mechanisms; in addition, the FT/TFL1 family of genes as an important potential breeding target is explored.