Transcriptional Regulatory Mechanism Of Differential Accumulation Of Carotenoids In The Flower Of Chrysanthemum ×Morifolium

Carotenoids,a group of important secondary metabolites that make flowers and fruits appear in diverse colors,are biosynthesized and storaged in all photosynthetic organisms,including algae and plants.Recently,significant progress has been made in carotenoid metabolism.However,the specific understanding on regulation mechanism controlling the expression of carotenoid metabolic genes remains extremely limited.Chrysanthemum ×morifolium,one of the top ten well-known traditional Chinese flowers,is a leading flower with applied value worldwide.Flower colour is the most important trait in chrysanthemum.Carotenoids,a subclass of terpenoids,make the petals of chrysanthemum appear yellow or orange.In this study,a set of flower color bud mutational cultivars(including ‘Jianliuxiang Pink’,‘Jianliuxiang White’,‘Jianliuxiang Yellow’ and ‘Jianliuxiang Red’)and an anemone-type chrysanthemum cultivar ‘Dong Li Fen Gui’ were collected.Then a series of physiological and molecular biological assays were performed to explore the genetic regulatory mechanism of carotenoid metabolism in chrysanthemum.The main results are described as follows:1.A pink colored chrysanthemum cultivar,‘Jianliuxiang Pink’,and its three bud sport mutants(including white,yellow and red color mutants,‘Jianliuxiang White’,‘Jianliuxiang Yellow’ and ‘Jianliuxiang Red’,respectively)were used as experimental materials to analyze the dynamic changes of carotenoid components and plastid ultrastructure at different developmental stages of ray florets.The carotenoid components and plastid ultrastructure of these four color cultivars in the early developmental stage of the chrysanthemum capitulum(S1)were almost identical,and the carotenoids mainly included violaxanthin,lutein and β-carotene,which existed in proplastids and immature chloroplasts.As capitulum developed,the chloroplasts in ‘Jianliuxiang White’ and‘Jianliuxiang Pink’ were degraded,and the protoplasts can not transform but rather formed vesicles that only accumulated trace amount of carotenoids.While the proplastids and chloroplasts in ‘Jianliuxiang Yellow’ and ‘Jianliuxiang Red’ were all transformed into chromoplasts,accumulating lutein as well as lutein’s isomer and derivatives.Using comparative transcriptomics combined with gene expression analysis,Cm Pg-1,Cm PAP10,and Cm PAP13,which were involved in chromoplast transformation,Cm LCYE,which was involved in carotenoid biosynthesis,and Cm CCD4a-2,which was involved in carotenoid degradation,were differentially expressed among these four chrysanthemum cultivars,and it showed that all these key structural genes affect the accumulation of carotenoids in the ray florets of chrysanthemum.In addition,nine transcription factors,Cm MYB305,Cmb ZIP61,Cmb HLH62,Cm AGL24,Cm RAD3,Cm NAC1,Cm RAP2-3,Cm MYB29 and Cm IAA,were screened using weighted gene co-expression correlation network analysis(WGCNA)combined with correlative analysis to determine whether they play an important role in carotenoid accumulation by regulating structural genes related to the carotenoid metabolism pathway and plastid development.2.Based on the previous transcriptome data combined with WGCNA analysis and homologous gene comparison,a total of 11 R2R3-MYB transcription factors that might be involved in the transcriptional regulation of carotenoid metabolism in the ray florets of chrysanthemum were screened out.Cm MYB21-1,which belonged to the S21 subfamily of the R2R3-MYBs and was closely related to the El RCP1,was regarded as the candidate transcription factor that played an important role in promoting carotenoid accumulation by dual-luciferase reporter assays.After transient overexpression of Cm MYB21-1,a large amount of carotenoids were accumulated in the ray floret of chrysanthemum,and the expression levels of carotenoid-related genes,including Cm DXS1,Cm GGPPS,Cm PSY2,Cm PDS,Cm LCYB,Cm LCYE,Cm CYP97C(Cm CHYE)and Cm PAPs(such as Cm Pg1,Cm PAP10,Cm PAP13,involved in chromoplast differentiation)were increased significantly,indicating that Cm MYB21-1 may simultaneously affect the synthesis and storage of carotenoids in the ray floret of chrysanthemum.Furthermore,yeast two-hybrid assay showed that Cm MYB21-1 could directly interact with Cm MYB6,which was regarded as the transcriptional activator that promoted the synthesis of anthocyanins in chrysanthemum.It was speculated that Cm MYB21-1 may indirectly affect the accumulation of anthocyanins in chrysanthemum through protein-protein interaction.3.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.Here,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 Cm CCD4a-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 Cm CCD4a-2 was used as bait to screen a chrysanthemum flower c DNA library and two transcription factors,Cm AP3 and Cm UIF1 were identified.Y2 H,Bi FC and Y3 H experiments demonstrated that these two TFs were connected by Cm PI to form Cm AP3-Cm PI-Cm UIF1 TF complex.This TF complex regulated carotenoid metabolism through activating the expression of Cm CCD4a-2 directly.Furthermore,a large number of target genes regulated directly by the Cm AP3-Cm PI-Cm UIF1 TF complex,including carotenoid biosynthetic genes,flavonoid biosynthetic genes and flower developmentrelated genes,were identified by DNA-affinity purification sequencing(DAP-seq),which indicated that the Cm AP3-Cm PI-Cm UIF1 TF complex might participate in multiple processes.Based on the above results,the molecular regulatory mechanism of carotenoid metabolism was preliminarily clarified in chrysanthemum.On the one hand,Cm Pg-1,Cm PAP10,and Cm PAP13,which were involved in chromoplast transformation,Cm LCYE,which was involved in carotenoid biosynthesis,and Cm CCD4a-2,which was involved in carotenoid degradation,affect carotenoid accumulation in the ray florets of chrysanthemum together.The R2R3 MYB transcription factor Cm MYB21-1,as an upstream transcription activator,can activate the expression of key structural genes to promote the biosynthesis and accumulation of carotenoids.On the other hand,carotenoid cleavage oxygenase Cm CCD4a-2 was the most critical structural gene that resulted in the distinct content of carotenoids accumulated in the ray and disc florets.Then a TF complex named Cm AP3-Cm PI-Cm UIF1 was identified and this TF complex regulated carotenoid metabolism through activating the expression of Cm CCD4a-2 directly,thereby different content of carotenoids was accumulated in these two types of florets.In this study,a series of key structural genes and upstream transcription factors,which affect the carotenoid metabolism in chrysanthemum,were excavated.Besides,the regulatory networks between these key genes were constructed.These findings expand our knowledge for the transcriptional regulation of carotenoid metabolism in plants and will be helpful to manipulating carotenoid accumulation in chrysanthemum.