Molecular Regulatory Mechanism Of Petal Coloration In Cornflower

Flower color is one of the most important traits in ornamental plants.Blue flower is extremely favored by consumers for its rareness as well as fantasy and is the key breeding goal in horticultural innovation.Pigments are key factors in deciding petal color,in which anthocyanins play the most important role.Generally,delphinidin derivatives facilitate bright blue petal colors.However,Chrysanthemum × morifolium Ramat.,one of the most important cut flowers worldwide,accumulates only cyanidin derivatives in its ray flowers and exhibits different degrees of red colors.Interestingly,cyanidin derivatives in Centaurea cyanus L.,species belonging to Asteraceae,make petal exhibit pure and bright blue color,suggesting cyanidin derivatives could also develop blue color in certain conditions.Previous studies showed there were supramolecular pigments composed of cyanidin derivatives,apigenin derivatives as well as metal ions in blue cornflower petals.However,its potential molecular regulatory mechanism remains unclear.There are abundant petal color variations in cornflower except for the commonly blue color,including white,pink,red,mauve and black colors,which make it an ideal material to explore potential mechanisms.On the one hand,the present study traced the petal color development by analysis its dynamic changes of pigments,metal element contents and transcripts in blue cornflower.Then,a series of molecular biology assays were conducted to explore the transcriptional regulation mechanism of key genes.The molecular mechanism involved in the blue coloration of cornflower was preliminarily revealed.On the other hand,six cornflower cultivars with different petal colors were collected.Based on the analysis of their pigments,the expression pattern and sequence character of key genes were further explored to speculate their flavonoid biosynthetic pathways and reveal the molecular mechanism of petal color variation among cornflower cultivars.The molecular regulatory mechanism of petal coloration in cornflower was preliminarily revealed combining those comparative analysises within and among cultivars.The main results were described as followings:(1)The dynamic color development happened in blue cornflower petals,which changed from white to violet and eventually to blue.The ultra-performance liquid chromatography coupled with mass spectrometry was applied to trace the pigment changes both qualitatively and quantitatively,and the results revealed that both blue and mauve regions accumulated similar apigenin derivatives,but the content of cyanidin derivatives in blue regions were nearly 2.5-folds higher than that in violet regions,suggesting the relative content ratio of different flavonoids may play an important role in the blue color development of cornflower.RNA-seq was performed using the white,violet and blue color regions on the same petal and 105,506 unigenes were obtained by de novo assembling.A total of 46 unigenes involved in the flavonoid biosynthesis were selected and unigenes involved in the anthocyanin biosynthesis showed the highest expression peak in violet regions,while expression patterns of unigenes involved in the apigenin biosynthesis decreased gradually along with petal color development.Five transcription factors,including four R2R3-Cc MYBs and one IIIf-Ccb HLH1,were screened out using bioinformatic analysis,and their expression levels increased with petal color development.In addition,eight differentially expressed genes related to metal ions were digged out including ferritin,ferrochelatase,vacuolar iron transporter,magnesium transporter,metallothionein and metal tolerance protein,and they were significantly highly expressed in the violet and blue regions and may be involved in the generation of blue supramolecular pigment in cornflower.(2)To explore the transcriptional regulation mechanism in cornflower,promoter sequences of three key biosynthetic genes including Cc F3 H,Cc DFR and Cc FNS were isolated,and the bioinformatic analysis indicated that there were multiple cis-elements recognized by MYB and b HLH proteins.The dual luciferase assay revealed that Cc MYB6-1 significantly upregulated the activity of Cc F3 H and Cc DFR promoters,and this activity was obviously enhanced when co-infiltrated with Ccb HLH1;Transient over-expression assay showed that the co-infiltration of Cc MYB6-1 and Ccb HLH1 significantly induced anthocyanin accumulation in tobacco leaves;Both yeast two-hybrid and bimolecular fluorescence complementary experiments indicated the protein-protein interaction between Cc MYB6-1 and Ccb HLH1.These results suggested that Cc MYB6-1 and Ccb HLH1 were two activators regulating the anthocyanin biosynthesis in cornflower.In addition,all five candidate transcription factors could not upregulate the activity of Cc FNS promoter,suggesting that the transcriptional regulation mechanism of co-pigment biosynthesis was different from that of anthocyanin biosynthesis.(3)Six cornflower cultivars with white,pink,red,blue,mauve and black colors were collected.Their petal color phenotypes were defined precisely by both RHSCC and colorimeter,followed by the isolation of pigments by ultra-performance liquid chromatography.A total of ten anthocyanin derivatives and eight co-pigments isolated and their structures were putatively identified by spectral characteristics combined with mass spectrum data.Except for white petals without any anthocyanins,both pink and red flowers contained pelargonidin derivatives,while blue,mauve and black petals accumulated cyanidin derivatives;Apigenin derivatives were widely accumulated in different cultivars;Flavonoids in cornflower were widely acylated with aliphatic acids mainly including malonic and succinic acids;Both the mauve and blue petals accumulated the same cyanidin derivatives,whereas the apigenin derivative in mauve petals lost the glucosylation and acylation modifications on the 4' position,leading to the speculation that the different modification of apigenin aglycon might cause their petal color variations;Flavonoid contents were analyzed by semi-quantitative method using standards,and the relative content of total anthocyanin to total co-pigments varied among cultivars,suggesting it might play an important role in petal coloration.The obtained results preliminarily illustrated the pigment basis of petal color variation among cornflower cultivars.(4)To explore the molecular mechanism involved in the branch pathway of flavonoid biosynthesis,the expression patterns of seventeen biosynthetic genes and five transcription factors were detected in six cornflower cultivars with different petal colors and their developmental stages,and the sequences of key genes were also isolated.The transcripts of key genes involved in anthocyanin biosynthesis,such as Cc F3 H,Cc F3'H,Cc DFR and Ccb HLH1 etc.,were undetectable in white petals.Further sequence analysis revealed that its Ccb HLH1 was mutated and lost the conserved motifs,which might be responsible for its anthocyanin absence;Cc F3'H in pink and red petals also had a mutation and lost several key motifs,which might account for their accumulation of pelargonidin derivatives rather than cyanidin derivatives;The expression abundances of most anthocyanin biosynthesis genes in red and black petals continuously increased with color development and were significantly higher than other cultivars,which was corresponding to their hyper-accumulation of anthocyanins;The expression patterns of both Cc FNS and Cc GT3 decreased with petal color development,and Cc GT3 was undetectable in both mauve and red petals.Previous pigment analysis showed the apigenin derivative in mauve and red petals lacked glucosylation modification on the 4' position.Therefore,we speculated that Cc GT3 was involoved in the glucosylation of apigenin.These results indicated that the branch pathways of flavonoid biosynthesis among cornflower cultivars were mainly caused by the mutation of biosynthetic genes or transcription factor.Based on the above results,the molecular regulatory mechanism of petal coloration was preliminarily clarified in cornflower.On the one hand,during the blue color development of cornflower petals,lower concentrations of cyanidin derivatives only facilitated the violet colors and higher ones made petal exhibit pure blue color under the same accumulation of apigenin derivatives.Namely,the relative content between cyanidin and apigenin derivatives played an important role in the blue petal coloration of cornflower.Anthocyanin biosynthesis was synergistically regulated by transcription factors Cc MYB6-1 and Ccb HLH1,which provided sufficient cyanidin derivatives for the generation of blue supramolecular pigment in cornflower.On the other hand,the petal color variation among cornflower cultivars was mainly caused by different accumulation of pigments,mainly including the pelargonidin,cyanidin and apigenin biosynthetic pathways.The mutation of Ccb HLH1 in white cultivar resulted in the loss of conserved motifs and the absence of anthocyanin accumulation.The mutation of Cc F3'H,a key gene involved in cyanidin biosynthesis,resulted in the pelargonidin biosynthesis in pink or red cornflowers.Both the mauve and blue cultivars contained the same cyanidin derivative,but the former accumulated an apigenin derivative losing the glucosylation and acylation modification on the 4'position caused by a loss of Cc GT3.Moreover,the hyper-accumulation of cyanidin derivatives accounted for the darker coloration in the black cultivar.The present study provides genetic resources for understanding the blue coloration mechanism based on cyanidin derivatives and further enriches the theoretical research on petal color variation in ornamental plants.