Dynamic changes in petal colour and flavonoid composition in blue cornflower
The pigmentation of cornflower petal occurs from top to bottom gradually. Interestingly, the initial colour was white when petals were embedded in the bracts, and then it changed to violet and finally be pure blue colour, while the three different colour regions coexisted in S2 (Fig. 1a). Quantitative description results showed significant differences among these colour regions on the same petal, with decreasing L* as well as increasing a*, |b*| and C* during colour development (Fig. 1b). UPLC-MS/MS was performed to detect the flavonoid profiling changes both qualitatively and quantitatively. In total, five flavonoids were isolated, including the detection of two anthocyanidin derivatives at 525 nm (Fig. 1d) and three flavone derivatives at 350 nm (Fig. 1e). Based on the characteristics determined by UV-vis absorption spectroscopy, mass spectrum data (Additional file 1: Table S1) and a previous study , peak a1 and a2 were putatively identified as cyanidin-3-O-malonyl-glucoside-5-O-glucoside (Cy3malonylG5G) and cyanidin-3-O-(6''-succinyl-glucoside)-5-O-glucoside (Cy3succinylG5G), respectively. Peak f1 was putatively assigned as luteolin-7-O-succinyl-glucoside (Lu7succinylG), while peak f2 and f3 sharing the same mass spectrum were identified as mutual isomers, namely apigenin-4'-O-(6''-O-malonyl-glucoside)-7-O-glucuronide (Ap4'malonylG7Gn) (Additional file 1: Table S1) . The quantitative analysis suggested that cyanidin accumulation started in the violet region and reached a maximum in the blue region by as high as 2.5-fold. Moreover, apigenin was initially produced in the white region and reached the highest level in the remaining two colour regions (Fig. 1c). The chemical structure formulas of the main accumulated flavonoids are also shown (Fig. 1f). These results revealed that the blue supramolecular complex in cornflower was generated in a gradual and stoichiometric way, namely, the co-existence of cyanidin and apigenin derivatives as well as their relative ratio were both essential in blue colour development.
Transcriptomic changes during petal colour development
To further explore the molecular mechanisms underlying blue colour development, transcriptome results were obtained from three colour regions (white, violet and blue) on the same petal in S2 with three biological replicates (Fig. 1a). A total of 91.74 G clean bases were obtained with a Q30 base percentage greater than 93%, and short reads were assembled into 105,506 unigenes with a mean length of 1260 bp, suggesting that the sequencing quality was sufficiently high to ensure further analysis (Additional file 2: Table S2). Gene expression was analysed by fragments per kilobase of transcript per million mapped reads (FPKM), and differentially expressed genes (DEGs) were identified among the three colour regions. In comparison to the white region, there were 10,296 and 5,249 DEGs in the blue vs white and violet vs white comparisons, respectively, while the DEGs decreased to 1,601 between the blue vs violet comparison (Fig. 2). This distribution of DEGs suggested that the adjoining colour regions possessed relatively similar life processes and metabolic activities. The GO enrichment analysis indicated that these three comparisons produced similar representations of GO terms that were abundantly enriched in the metabolic process (GO:0008152), single-organism metabolic process (GO:0044710) and oxidation-reduction process (GO:0055114) within the biological process, in the external encapsulating structure (GO:0030312), cell wall (GO:0005618) and fatty acid synthase complex (GO:0005835) within the cellular component, as well as in the catalytic activity (GO:0003824), transferase activity (GO:0016740) and oxidoreductase activity (GO:0016491) within the molecular function under the corrected p-value <10-4 (Additional file 3: Fig. S1; Additional file 4: Table S3). The KEGG pathways, including phenylpropanoid biosynthesis (ko00940) and flavonoid biosynthesis (ko00941), were both significantly enriched in three comparisons. Moreover, starch and sucrose metabolism (ko00500) were the most abundantly enriched pathways when comparing the blue and violet regions to the white region (Additional file 3: Fig. S1; Additional file 5: Table S4). These results provide a global view on the potential life processes and metabolic activities during blue colour development.
Expression patterns of flavonoid biosynthetic genes during petal colour development
Combining the UPLC-MS/MS and transcriptome results, the pigment components and related metabolism played important roles in blue colour development, thereby leading to the excavation of structural genes involved in flavonoid biosynthesis in the combined functional annotations. A total of 46 unigenes were selected as the study focus (Additional file 6: Table S5). Subsequently, the transcriptional profiles showed that the key unigene involved in flavone biosynthesis, FNS, was expressed the highest level in the white region and decreased gradually along with further petal colour development to violet and blue colours (Fig. 3a-b). In contrast, the anthocyanin generation genes, including both early biosynthetic genes (F3H, F3'H) and late ones (DFR, ANS, GT, AT), showed the highest expression peak in the violet region (Fig. 3a&c). These results suggested that the highest gene expression peaks of apigenin and cyanidin biosynthesis occurred earlier than their accumulation peak. To further verify the credibility of the transcriptome data, eighteen DEGs were subjected to qRT-PCR, and the correlation coefficient between them was as high as 0.82, suggesting that the transcriptome was reliable (Fig. 3d).
TFs involved in flavonoid biosynthesis in cornflower
The above results indicated that flavonoid accumulation and gene expression occurred in a gradual and precise manner, which was potentially regulated by upstream transcription factors (TFs). Thus, we acquired a global view of the MYBs and bHLHs obtained in the transcriptome database to further clarify the regulatory mechanisms. A total of 29 and 126 R2R3-MYBs in cornflower and Arabidopsis, respectively, were used to construct a phylogenetic tree (Fig. 4a). MYBs in Sg4, Sg5, Sg6 and Sg7 have been reported to play important roles in flavonoid biosynthesis . In our study, there were two CcMYBs in Sg4 (Cluster 7159.48611 and Cluster-7159.50459, renamed as CcMYB4-1 and CcMYB4-2, respectively) and two CcMYBs in Sg6 (Cluster 7159.49786 and Cluster-7159.43444, renamed as CcMYB6-1 and CcMYB6-2, respectively). All four MYBs contained the typical R2 and R3 conserved domains by sequence analysis (Fig. 4c). Additionally, a phylogenetic tree of bHLHs, including 19 CcbHLHs and 145 AtbHLHs, was also constructed using the conserved domains (Fig. 4b). The subfamilies were labelled following the Arabidopsis bHLH group nomenclature . Four typical conserved bHLH regions were detected in cornflower bHLH, namely one basic region, two helix regions and one loop region (Fig. 4d). Based on previous study, bHLH proteins of the Ⅲf subgroups (TT8, GL3, EGL3 etc.) can interact with R2R3-MYBs and are involved in flavonoid biosynthesis [22, 23]. Therefore, the unigene of cluster-7159.49153 integrated with Ⅲf subclades in Arabidopsis was selected as a potential regulator involved in flavonoid biosynthesis and designated CcbHLH1. Network interaction analysis has recently been developed as a powerful method to predict gene function. Further, the function of five candidate genes was predicted by using the online software STRING 11.0. All of them were highly homologous to the flavonoid biosynthetic genes in Arabidopsis, such as AtMYB4, AtMYB90, AtMYB114 and TT8 (Additional file 7: Fig. S2; Additional file 8: Table S6). Moreover, qRT-PCR analysis showed continuously increased expression levels of five candidate genes with petal colour development from white to violet and finally to blue colour (Fig. 5a).
Identification of TFs regulating cyanidin biosynthesis
In comparison to the violet region, the blue region possessed similar apigenin but accumulated 2.5-fold higher cyanidins, suggesting that an appropriate accumulation of cyanidins played an important role in blue colour development in cornflower, which led to further exploration of the potential regulatory mechanisms. Flavanone-3-hydroxylase (F3H) is the first key enzyme determining the cyanidin flux, and dihydroflavonol 4-reductase (DFR) catalysed the transformation of dihydroquercetin to leucocyanidin, both of which play crucial roles in cyanidin biosynthesis . Therefore, the promoters of CcF3H and CcDFR were obtained by genome walking technology with a length of 1644 bp and 1510 bp, respectively, to clarify the possible regulatory roles of CcMYBs and CcbHLH1 in cyanidin biosynthesis. The cis-elements were widely located in two promoters, such as MYBCORE(CNGTTR), MYBPLANT(MACCWAMC), and MYBPZM(CCWACC) recognized by MYB protein and EBOXBNNAPA(CANNTG) recognized by bHLH protein (Fig. 5b). Furthermore, a dual luciferase assay was conducted to explore the in vivo regulatory roles. The results showed that only CcMYB6-1 was able to trans-activate the CcF3H and CcDFR promoters compared with the empty vector, with approximately 13-fold and 32-fold induction, respectively, while single CcMYB6-2, CcMYB4-1, CcMYB4-2 and CcbHLH1 were unable to induce any promoter activity (Fig. 5c). MYB and bHLH transcription factors usually form a protein complex to regulate anthocyanin biosynthesis. In the presence of CcMYB6-1 and CcbHLH1, the activity of the CcF3H and CcDFR promoters was stimulated more than 17-fold and 46-fold, respectively. However, CcbHLH1 co-expression with CcMYB6-2, CcMYB4-1, and CcMYB4-2 was still unable to upregulate the activity of the two promoters (Fig. 5c).
A transient expression assay was further conducted in tobacco leaves to effectively verify the roles of CcMYBs and CcbHLH1 in regulating anthocyanin biosynthesis. CcMYB6-2, CcMYB4-1 and CcMYB4-2 were unable to induce anthocyanin biosynthesis either alone or co-expressed with CcbHLH1 (data not shown), which was consistent with the results of dual luciferase assay (Fig. 5c). In contrast, there were visible red spots on tobacco leaves infiltrated with CcMYB6-1. Moreover, co-expression of CcMYB6-1 and CcbHLH1 triggered a clearly larger red area (Fig. 5d), the anthocyanin content of which was almost five times higher than the level accumulated in tobacco leaves infiltrated with CcMYB6-1 and SK (Fig. 5e). This result suggested that CcMYB6-1 and CcbHLH1 possessed a positive ability to regulate anthocyanin biosynthesis.
Excavation of metal ion related genes
According to a previous study, the blue supramolecular pigment in cornflower is composed of six anthocyanins, six flavones, one Fe3+, one Mg2+ and two Ca2+ . Pigments synthesized in the endoplasmic reticulum were further transported into the vacuole to be protected from oxidation and subsequent loss of colour . Therefore, the metal ions should also be transported into the vacuole to chelate flavonoids. We further screened unigenes with different expression levels among the three colour regions in the transcriptome database. A total of eight DEGs potentially involved in metal ion transport, storage, chelation and tolerance were screened out, including one each of ferritin, ferrochelatase, vacuolar iron transporter, and magnesium transporter and two each of metallothionein and metal tolerance protein (Fig. 6a). The expression levels of these genes mostly increased with petal colour development from white to violet and finally to blue. Notably, the expression level of metallothionein was at least 18-fold higher than the other unigenes (Fig. 6b). Pearson’s correlation analysis showed that the expression patterns of ferrochelatase, magnesium transporter and metallothionein were significantly correlated with |b*| (r>0.9, p<0.001***), an important parameter representing the degree of blue, and the expression levels of ferritin as well as metal tolerance protein also exhibited a strong correlation (r>0.8, p<0.01**) (Additional file 9: Table S7). The concrete functions of these candidate genes in the generation of blue supramolecular pigment remain to be further explored.