Functional analysis of the ScAG and ScAGL11 MADS-box transcription factors for anthocyanin biosynthesis and bicolour pattern formation in Senecio cruentus ray florets.

Cineraria (Senecio cruentus) is an ornamental plant with pure colour and bicolour cultivars, widely used for landscaping. Anthocyanin biosynthesis influences coloration patterns in cineraria. However, how anthocyanins accumulate and distribute in cineraria is poorly understood. This study investigated the molecular mechanisms underlying anthocyanin biosynthesis and bicolour formation in cineraria using pure colour and bicolour cultivars. Transcriptome and gene expression analysis showed that five genes, ScCHS2, ScF3H1, ScDFR3, ScANS, and ScbHLH17, were inhibited in the white cultivar and colourless regions of bicolour cultivars. In contrast, two MADS-box genes, ScAG and ScAGL11, showed significantly higher expression in the colourless regions of bicolour cultivars. ScAG and ScAGL11 were localized in the nucleus and co-expressed with the bicolour trait. Further functional analysis verified that ScAG inhibits anthocyanin accumulation in tobacco (Nicotiana tabacum). However, virus-induced gene silencing (VIGS) experiments showed that silencing of ScAG and ScAGL11 increases anthocyanin content in cineraria leaves. Similar results were observed when ScAG and ScAGL11 were silenced in the cineraria capitulum, accompanied by the smaller size of the colourless region, specifically in the ScAG/ScAGL11-silenced plants. The expression of ScCHS2, ScDFR3, and ScF3H1 increased in silenced cineraria leaves and capitulum. Furthermore, yeast two-hybrid and bimolecular fluorescence complementation experiments demonstrated that ScAG interacts with ScAGL11. Moreover, ScAG directly inhibited the transcription of ScF3H1 while ScAGL11 inhibited ScDFR3 expression by binding to their promoters separately. The findings reported herein indicate that ScAG and ScAGL11 negatively regulate anthocyanin biosynthesis in cineraria ray florets, and their differential expression in ray florets influences the bicolour pattern appearance.

ScGST3 and multiple R2R3-MYB transcription factors function in anthocyanin accumulation in Senecio cruentus.

Anthocyanins are important flavonoid pigments involved in the colouring of flowers and fruits. They are synthesized on the cytoplasmic surface of the endoplasmic reticulum and transported into the vacuole for storage. Previous reports have suggested that glutathione S-transferase (GST) is involved in anthocyanin transport. However, due to the limitation of plant materials, most GSTs only participate in the cyanidin or delphinidin transport pathway. Here, an anthocyanin-related GST, ScGST3, was identified from the transcriptome of cineraria. The expression pattern of ScGST3 was highly consistent with anthocyanin accumulation in ray florets. Molecular complementation of Arabidopsis tt19 indicated that the overexpression of ScGST3 restores the anthocyanin-deficient phenotype of the mutant. Virus-induced gene silencing (VIGS) of ScGST3 in carmine and blue cineraria leaves could inhibit anthocyanin accumulation, further confirming the function of ScGST3 in anthocyanin accumulation. In vitro assays showed that ScGST3 increases the water solubility of cyanidin-3-O-glucoside (C3G) and delphinidin-3-O-glucosid (D3G). In addition, we also identified two anthocyanin-related MYB transcription factors, ScMYB3 and ScMYB6. The expression pattern of these two genes was also highly consistent with anthocyanin accumulation. Faded abaxial leaf phenotypes were observed after the silencing of ScMYB3 and ScMYB6, and the expression levels of partial structural genes were repressed. Based on the results from dual-luciferase assays and yeast one-hybrid assays, ScMYB3 can activate the promoter of ScGST3. Collectively, the transcription of ScGST3 is regulated by ScMYB3, which plays an important role in the transport of C3G and D3G in cineraria.

Isolation and Functional Analysis of Genes Involved in Polyacylated Anthocyanin Biosynthesis in Blue Senecio cruentus.

Polyacylated anthocyanins with multiple glycosyl and aromatic acyl groups tend to make flowers display bright and stable blue colours. However, there are few studies on the isolation and functional characterization of genes involved in the polyacylated anthocyanin biosynthesis mechanism, which limits the molecular breeding of truly blue flowers. Senecio cruentus is an important potted ornamental plant, and its blue flowers contain 3',7-polyacylated delphinidin-type anthocyanins that are not reported in any other plants, suggesting that it harbours abundant gene resources for the molecular breeding of blue flowers. In this study, using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis of blue, carmine and white colours of cineraria cultivars "Venezia" (named VeB, VeC, and VeW, respectively), we found that 3',7-polyacylated anthocyanin, cinerarin, was the main pigment component that determined the blue colour of ray florets of cineraria. Based on the transcriptome sequencing and differential gene expression (DEG) analysis combined with RT- and qRT-PCR, we found two genes encoding uridine diphosphate glycosyltransferase, named ScUGT1 and ScUGT4; two genes encoding acyl-glucoside-dependent glucosyltransferases which belong to glycoside hydrolase family 1 (GH1), named ScAGGT11 and ScAGGT12; one gene encoding serine carboxypeptidase-like acyltransferase ScSCPL2; and two MYB transcriptional factor genes ScMYB2 and ScMYB4, that were specifically highly expressed in the ray florets of VeB, which indicated that these genes may be involved in cinerarin biosynthesis. The function of ScSCPL2 was analysed by virus-induced gene silencing (VIGS) in cineraria leaves combined with HPLC-MS/MS. ScSCPL2 mainly participated in the 3' and 7-position acylation of cinerarin. These results will provide new insight into the molecular basis of the polyacylated anthocyanin biosynthesis mechanism in higher plants and are of great significance for blue flower molecular breeding of ornamental plants.

Establishment of virus-induced gene silencing system and functional analysis of ScbHLH17 in Senecio cruentus.

Virus-induced gene silencing (VIGS) is a technology for rapid gene functional analysis that depends on the degradation of viral RNA and is part of the natural defense mechanism in plants. Senecio cruentus is an important Compositae ornamental species that is plentiful and available in a variety of colors and has a typical blue variety that is rare in Compositae. These advantages make it a good material for studying the anthocyanin biosynthesis and blue flower formation mechanism. With the development of gene sequencing technology, the functions of many candidate genes that may be involved in anthocyanin biosynthesis in S. cruentus need to be identified. However, a stable and rapid genetic transformation system of S. cruentus is still lacking. Here, we screened two cultivars, 'Venezia' and 'Jseter', selected ScPDS and ScANS as test genes, and investigated the effect of developmental periods, bacterial cell concentrations and infection methods on gene silencing efficiency. The results showed that the silencing efficiency of S. cruentus leaves was low (13%), and it was less affected by the parameters. However, the transcription factor gene ScbHLH17 was still silenced by VIGS, which resulted in the loss of anthocyanin accumulation in leaves, and the expression levels of anthocyanin biosynthesis pathway (ABP) structural genes, including ScCHI, ScDFR3 and ScANS, were decreased significantly. The result proved that ScbHLH17 was an important transcription factor that regulated flower color formation in S. cruentus. In addition, ScANS-silencing phenotypes were observed in S. cruentus capitulum by vacuum-infiltrating S1 stage buds for 10 min after scape injection. In general, the present study provided an important technical support for the study of anthocyanin metabolism pathways in S. cruentus.