The transcription factor complex CmAP3-CmPI-CmUIF1 modulates carotenoid metabolism by directly regulating carotenogenic gene CmCCD4a-2 in chrysanthemum.

Carotenoids are one of the most important pigments for the coloring in many plants, fruits and flowers. Recently, significant progress has been made in carotenoid metabolism. However, the specific understanding on transcriptional regulation controlling the expression of carotenoid metabolic genes remains extremely limited. Anemone-type chrysanthemum, as a special group of chrysanthemum cultivars, contain elongated disc florets in capitulum, which usually appear in different colors compared with the ray florets since accumulating distinct content of carotenoids. In this study, the carotenoid composition and content of the ray and disc florets of an anemone-type chrysanthemum cultivar 'Dong Li Fen Gui' were analyzed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and the key structural gene CmCCD4a-2, of which differential expression resulted in the distinct content of carotenoids accumulated in these two types of florets, was identified. Then the promoter sequence of CmCCD4a-2 was used as bait to screen a chrysanthemum flower cDNA library and two transcription factors, CmAP3 and CmUIF1 were identified. Y2H, BiFC and Y3H experiments demonstrated that these two TFs were connected by CmPI to form CmAP3-CmPI-CmUIF1 TF complex. This TF complex regulated carotenoid metabolism through activating the expression of CmCCD4a-2 directly. Furthermore, a large number of target genes regulated directly by the CmAP3-CmPI-CmUIF1 TF complex, including carotenoid biosynthetic genes, flavonoid biosynthetic genes and flower development-related genes, were identified by DNA-affinity purification sequencing (DAP-seq), which indicated that the CmAP3-CmPI-CmUIF1 TF complex might participate in multiple processes. These findings expand our knowledge for the transcriptional regulation of carotenoid metabolism in plants and will be helpful to manipulating carotenoid accumulation in chrysanthemum.