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Virus-induced gene silencing (VIGS) is a genetic tool used to assess gene function. Tobacco rattle virus (TRV) is a VIGS vector commonly used to induce endogenous gene silencing in plants. However, there is no VIGS system established for Centaurea spp. We evaluated the effectiveness of a TRV-based VIGS system using phytoene desaturase (PDS) as a reporter gene in Centaurea cyanus. Three methods including pressure-, vacuum- and apical meristem-infiltration were tested to infect C. cyanus seedlings. Photobleached leaves were only obtained using apical meristem-infiltration after a 14 d treatment. The CcPDS transcripts in photobleached leaves were significantly reduced compared with that in green leaves treated with empty TRV. Four C. cyanus cultivars were tested to detect their VIGS responses, and ‘Dwarf Tom Pouce Blue’ was the most sensitive. The agro-infiltration condition was optimized by screening for the optimal seedling stage as well as the optimum Agrobacterium density for efficient silencing. Seedlings with four true leaves and infiltration with an Agrobacterium density of OD600 0.5 were optimal conditions to obtain more photobleached leaves and more intense photobleached phenotype. The results demonstrated the feasibility of TRV-based VIGS for functional analysis of genes in C. cyanus.

Anthocyanins in cornflower (Centaurea cyanus) is catalysed by a set of biosynthesis genes, however, the potential mechanism of transcriptional regulation remains unclear. In the present study, we traced the dynamic changes of petal colour development from white to violet and finally to blue on the same petal in cornflower. Pigment analysis showed that anthocyanin accumulation dramatically increased with petal colour development. Subsequently, nine libraries from above three colour regions were constructed for RNA-seq and 105,506 unigenes were obtained by de novo assembling. The differentially expressed genes among three colour regions were significantly enriched in the phenylpropanoid biosynthesis and flavonoid biosynthesis pathways, leading to the excavation and analysis of 46 biosynthesis genes involved in this process. Furthermore, four R2R3-CcMYBs clustered into subgroup 4 or subgroup 6 and one CcbHLH1 clustered into IIIf subgroup were screened out by phylogenetic analysis with Arabidopsis homologues. The promoters of flavanone 3-hydroxylase (CcF3H) and dihydroflavonol 4-reductase (CcDFR) were further isolated to investigate upstream regulation mechanism. CcMYB6-1 significantly upregulated the activity of above two promoters and stimulated anthocyanin accumulation by dual luciferase assay and transient expression in tobacco leaves, and its activity was obviously enhanced when co-infiltrated with CcbHLH1. Moreover, both yeast two-hybrid and bimolecular fluorescence complementation assays indicated the protein-protein interaction between these two activators. Based on these obtained results, it reveals that CcMYB6-1 and CcbHLH1 are two novel transcription factors synergistically involved in regulating anthocyanin biosynthesis. This study provides insights into the regulatory mechanism of anthocyanin accumulation in cornflower.

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.

The variation of flower color of chrysanthemum (Chrysanthemum×morifolium) is extremely rich, and carotenoids, which are mainly stored in the plastid, are important pigments that determine the color of chrysanthemum. However, the genetic regulation of the carotenoid metabolism pathway in this species still remains unclear. In this study, a pink 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. We found that the carotenoid components and plastid ultrastructure of the 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 exist in proplastids and immature chloroplasts. With the development of capitulum, the chloroplasts in 'Jianliuxiang White' and 'Jianliuxiang Pink' were degraded, and the protoplasts did not transform but rather formed vesicles that accumulated trace amounts of carotenoids. The proplastids and chloroplasts in 'Jianliuxiang Yellow' and 'Jianliuxiang Red' were all transformed into chromoplasts and consist of lutein as well as lutein's isomer and derivatives. Using comparative transcriptomics combined with gene expression analysis, we found that CmPg-1, CmPAP10, and CmPAP13, which were involved in chromoplast transformation, CmLCYE, which was involved in carotenoid biosynthesis, and CmCCD4a-2, which was involved in carotenoid degradation, were differentially expressed between four cultivars, and these key genes therefore should affect the accumulation of carotenoids in chrysanthemum. In addition, six transcription factors, CmMYB305, CmMYB29, CmRAD3, CmbZIP61, CmAGL24, CmNAC1, 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. This study analyzed dynamic changes of carotenoid components and plastid ultrastructure of the four bud mutation cultivars of chrysanthemum and identified structural genes and transcription factors that may be involved in carotenoid accumulation. The above results laid a solid foundation for further analysis of the regulatory mechanism of the carotenoid biosynthesis pathway in chrysanthemum.

The understanding of capitulum morphogenesis is critical for modification of chrysanthemum flower type. Previous studies showed candidate genes which involved in the inflorescence architecture, such as PIN-FORMED 1 (PIN1), AUXIN RESPONSE FACTOR 5 (ARF5) and LEAFY (LFY), acted dominant role during early flower development. However, it is not known whether those candidate genes play parts in the capitulum morphogenesis. In the present study, floral morphogenesis and the corresponding external morphological changes in Chrysanthemum lavandulifolium were observed using scanning electron microscope and light microscope technology. Capitulum morphogenesis showed that ray florets primordia initiated later than the outermost disc florets primordia at the middle stage of florets primordia differentiation (stage 6), breaking the acropetal initiation of organ primordia on capitulum. Organogenesis of disc and ray florets showed that the early generation of florets corollas primordia was the key differentiation stages, in which disc and ray florets fused with actinomorphic and zygomorphic corollas, respectively. Furthermore, the expression patterns of inflorescence architecture related genes were detected at key stages of capitulum morphogenesis as well as the different organs on capitulum using quantitative real time PCR (qRT-PCR). The transcripts of ClARF5 specially increased at stage 5 and stage 6, as well as in ray florets corollas. The results indicated that ClARF5 might be responsible for the orientation and initiation of disc and ray florets during early capitulum morphogenesis. Studies on the capitulum morphogenesis of C. lavandulifolium not only bring molecular insights into capitulum morphogenesis, but also lay foundation for the flower type-oriented modification in cultivated chrysanthemums.

Flower type is an important quality trait of ornamental plants. Mastering genetic variation in flower types is of practical significance for breeding improved flower types. Chrysanthemum (non-anemone type) flowers consist of several elements; both ray floret shape and the relative number of ray florets (RNRF) on the capitulum are very important factors. However, until recently, few reports have standardized chrysanthemum flower type evaluations, hindering the development of genetic studies of chrysanthemum flower types. In this study, as an important index of ray floret shape, the corolla tube merged degree (CTMD) was defined as the corolla tube length/ray floret length (CTL/RFL). In addition, the number of ray florets/number of florets (NRF/NF) was used to describe the RNRF on the capitulum. Major gene and polygene mixed inheritance analyses were conducted based on 2 F1 populations from 2 pairs of parents that strongly differed in the CTMD and RNRF. ANOVA revealed that all traits strongly significantly differed among the different hybrids, and correlation analysis revealed that the CTMD and RNRF might have evolved independently. Additionally, according to the Q-mode cluster analysis results, we divided the RNRF into 4 levels: single, semi-double I, semi-double II and double. Genetic analysis revealed that both the CTMD and RNRF could be described by a B-2 genetic model via two additive-dominance major genes. In addition, the heritability of the major genes for these traits was greater than 50%, indicating that the CTMD and RNRF were controlled mainly by genetic factors. These results will provide a new theoretical basis for further improvement and breeding of chrysanthemum flower types.

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.

Flower color, which varies widely in potted multiflora chrysanthemum, is an extremely important character for classification. A quantitative definition of the flower color of potted multiflora chrysanthemums and the establishment of a standard color classification system may help us to evaluate the germplasm resources effectively. In this study, 273 potted multiflora chrysanthemum advanced lines were selected as materials. First, color classification results were compared between two methods: one is a combination of CIELAB and Munsell color systems, and the other one applying Q clustering analysis based on the CIELAB color system. We then divided the 273 lines into six color groups. The composition and content of anthocyanins and carotenoids in the different color advanced lines were identified by HPLC-MS. Two derivatives of cyanidin, cyanidin-3-O-(6″-O-malonyl-glucoside) and cyanidin-3-O-(3″,6″-O-dimalonyl-β-glucopyanoside), were identified in the pink, purple, orange-brown, and red lines. The carotenoids, primarily consisting of violanthin, z-violanthin, 9′z-lutein 5,6-epoxide, lutein, α-carotene, β-carotene, and 9z-β-carotene, were identified in all chrysanthemum advanced lines. Additionally, a*, represents the green-red component with green in the negative direction and red in the positive direction, was strongly positively correlated with cyanidin-3-O-(6″-O-malonyl-glucoside) (r = 0.884, P

Chrysanthemum (Chrysanthemum × morifolium) is an important horticultural crop with great ornamental and economic value. Most chrysanthemum cultivars are self-incompatible, which limits the creation of pure chrysanthemum lines which are the material basis for breeding and theoretical research. Here, artificial self-pollination and natural cross-pollination were used to analyze the self-compatibility (SC) and cross-compatibility (CC) of 85 chrysanthemums cultivated by our research group. By conducting a phenotypic study, observing of pollen germination in vitro and in vivo and gene expression analyses, we explored the reasons for the self-compatibility of chrysanthemum and identified self-compatible mutants. It was found that there were wide variations in self-compatibility among different chrysanthemum cultivars and the SC index ranged from 0 to 5.64, which could classify the chrysanthemum cultivars into three types: self-compatibility (SC index ≥ 1), self-incompatibility (SI, 1 > SC index > 0) and high self-incompatibility (SC index = 0). Seven chrysanthemum cultivars with self-pollinated progenies (‘Hongguan’, K183−8, A22, B12, D87, D76 and S8) were selected, and the pollen germination rates in vitro and in vivo were not significantly related to SC. The pollen tube of ‘Hongguan’ could pass through its own stigma and grow toward the embryo sac along the style, and there was no obvious inbreeding depression in self-pollinated progenies of ‘Hongguan’. In addition, we found that one S-locus receptor kinase (SRK), two S-locus cysteine-rich protein (SCR), two exocyst complex component EXo70A1 (Exo70A1) and one thioredoxin h-like 1 (THL1) were important for regulating self-compatibility of C. × morifolium. Our results not only laid a material foundation for creating pure lines through continuous self-pollination and analyzing the regulation mechanism underlying SC variation but also provided the possibility for theoretical research on the important ornamental traits of chrysanthemums in the future.

Cryptochrome (CRY), a vital photoreceptor which mediates light signals, controls photomorphogenesis in higher plants. However, the function of CRY in mediating light to regulate growth and development of ornamental plants is still unclear. In this study, we identified two CRY1 homologous genes, ClCRY1a and ClCRY1b, from Chrysanthemum lavandulifolium, a diploid wild chrysanthemum species. Expression analysis demonstrated that these two ClCRY1 genes showed the highest expression levels in seedlings leaves that were transferred to short day (SD) conditions for eight days. ClCRY1a was expressed at a higher level in the dark phase of SD, while ClCRY1b was expressed more highly in SD than that in long day (LD) conditions. Overexpression of either ClCRY1a or ClCRY1b in wild-type (WT) Arabidopsis resulted in early flowering under both LD and SD. The expression levels of GIGANTEA (GI) and FLOWERING LOCUS T (FT) were significantly up-regulated in ClCRY1a overexpressors under both LD and SD. Moreover, the transcript levels of GI, CONSTANS (CO) and FT were markedly increased in ClCRY1b overexpressors under LD; nevertheless, only the expression levels of CO and FT were up-regulated under SD. Taken together, the above results indicated that these two ClCRY1 genes could regulate flowering time via different pathways in C. lavandulifolium under LD and SD conditions. Our results provided evidence for the role of ClCRY1s in controlling photomorphogenesis and laid a foundation for further study on the molecular mechanism of ClCRYs mediating light signals to control floral transition.

Light is one of the key environmental factors that affect anthocyanin biosynthesis. However, the underlying molecular mechanism remains unclear, and many problems regarding phenotypic change and corresponding gene regulation have not been solved. In the present study, comparative analyses of light-induced anthocyanin accumulation and gene expression between the ray florets and leaves were performed in Chrysanthemum × morifolium 'Purple Reagan'. After contrasting the variations in the flower color phenotype and relative pigment content, as well as expression patterns of structural and regulator genes responsible for anthocyanin biosynthesis and photoreceptor between different plant organs under light and dark conditions, we concluded that (1) both the capitulum and foliage are key organs responding to light for chrysanthemum coloration; (2) compared with flavones, shading makes a greater decrease on the anthocyanins accumulation; (3) most of the structural and regulatory genes in the light-induced anthocyanin pathway specifically express in the ray florets; and (4) CmCHS, CmF3H, CmF3'H, CmANS, CmDFR, Cm3GT, CmMYB5-1, CmMYB6, CmMYB7-1, CmbHLH24, CmCOP1 and CmHY5 are key genes for light-induced anthocyanin biosynthesis in chrysanthemum ray florets, while on the transcriptional level, the expressions of CmPHYA, CmPHYB, CmCRY1a, CmCRY1b and CmCRY2 are insignificantly changed. Moreover, the inferred comprehensive effect of multiple signals on the accumulation of anthocyanins and transmission channel of light signal that exist between the leaves and ray florets were further discussed. These results further our understanding of the relationship between the gene expression and light-induced anthocyanin biosynthesis, and lay foundations for the promotion of the molecular breeding of novel flower colors in chrysanthemums.

In Arabidopsis, circadian clock genes play important roles in photoperiod pathway by regulating the daytime expression of CONSTANS (CO), but related reports for chrysanthemum are notably limited. In this study, we isolated eleven circadian clock genes, which lie in the three interconnected negative and positive feedback loops in a wild diploid chrysanthemum, Chrysanthemum lavandulifolium. With the exception of ClELF3, ClPRR1 and ClPRR73, most of the circadian clock genes are expressed more highly in leaves than in other tested tissues. The diurnal rhythms of these circadian clock genes are similar to those of their homologs in Arabidopsis. ClELF3 and ClZTL are constitutively expressed at all time points in both assessed photoperiods. The expression succession from morning to night of the PSEUDO RESPONSE REGULATOR (PRR) gene family occurs in the order ClPRR73/ClPRR37, ClPRR5, and then ClPRR1. ClLHY is expressed during the dawn period, and ClGIs is expressed during the dusk period. The peak expression levels of ClFKF1 and ClGIs are synchronous in the inductive photoperiod. However, in the non-inductive night break (NB) condition or non-24 h photoperiod, the peak expression level of ClFKF1 is significantly changed, indicating that ClFKF1 itself or the synchronous expression of ClFKF1 and ClGIs might be essential to initiate the flowering of C. lavandulifolium. This study provides the first extensive evaluation of circadian clock genes, and it presents a useful foundation for dissecting the functions of circadian clock genes in C. lavandulifolium.

Proline plays a significant role in plant resistance to abiotic stresses, and its level is determined by a combination of synthesis, catabolism and transport. The primary proteins involved are Δ(1)-pyrroline-5-carboxylate synthetase (P5CS), proline dehydrogenase (PDH) and proline transporter (ProT). To utilise proline metabolism to improve the stress resistance of Chrysanthemum×morifolium, we isolated two P5CS-homologous genes (ClP5CS1 and ClP5CS2), one PDH gene (ClPDH) and four ProT-homologous genes (ClProT1-4) (GenBANK accession numbers: KF743136-KF743142) from Chrysanthemum lavandulifolium, which is closely related to chrysanthemums and exhibits strong resistance to stresses. Expression analysis of these genes in different organs and under various stresses indicated that ClP5CSs showed substantial constitutive expression, while ClPDH was only strongly expressed in the capitulum and was inhibited under most stresses. The expression patterns of four ClProT genes presented characteristics of organ specificity and disparity under stresses. Above all, the expression of ClProT2 was restricted to above-ground organs, especially strong in the capitulum and could be obviously induced by various stress conditions. Promoters of ClPDH and ClProTs contained many cis-acting regulatory elements involved in stress responses and plant growth and development. High levels of free proline were found in flower buds, the capitulum under the non-stress condition and later periods of stress conditions except cold treatment. Interestingly, organ specificity and disparity also exist in the level of free proline under different stress conditions. Our study indicates that ClProTs play significant roles in proline accumulation and stress responses, and that ClProT2 could be used to genetically modify the stress resistance of chrysanthemums. In addition, proline metabolism might be closely related to plant flowering and floral development.

AP1/FUL-like genes encode class-specific MADS box transcription factors in angiosperms and play important roles in the plant floral transition and floral organ identity. Chrysanthemum lavandulifolium (Fisch. ex Trautv.) Makino is one of the original species of Chrysanthemum. Understanding the function of AP1/FUL-like genes in C. lavandulifolium provide molecular-level insight into the flowering mechanism of Chrysanthemum. Here, an AP1/FUL-like gene was isolated from C. lavandulifolium. Sequence and phylogenetic analyses indicated that this gene, ClM8, belongs to the euFUL clade of the AP1/FUL-like genes. We observed different expression levels of ClM8 in C. lavandulifolium roots, stems, fruits and leaves and high expression in the flowers. A subcellular localisation assay showed that ClM8 is localised to the nucleus in vivo. The over-expression of ClM8 in Arabidopsis resulted in altered flowering time and inflorescence structure. These aberrant, transgenic Arabidopsis plants showed early flowering following the production of four to six small rosette leaves, lateral flowers and a composite terminal flower. The terminal flower consisted of two or four pistils surrounded by an abnormal number of sepals, petals and stamens. In addition, the morphology of the style of 35S::ClM8 plants silique differed from wild type, appearing thinner and longer. Further investigation suggested that the expression levels of FLOWERING LOCUS T (FT), LEAFY (LFY) and APETALA1 (AP1) were apparently increased in ClM8-transgenic Arabidopsis. Thus, we speculate that ClM8 promoted flowering in Arabidopsis due to the upregulation of FT. Taken together, our results suggest that ClM8 is involved in flowering induction and floral development that might also play a role in the development of fruits and leaves.

Cineraria (Senecio cruentus Masson ex L’Herit) is a groundsel perennial herbaceous plant that has a variety of colors and is an ideal model used to research the mechanism of flower color variation. Anthocyanin is the major component that determines the flower color, and studying the regulation of endogenous anthocyanin biosynthesis-related genes is important for understanding the molecular mechanism of flower color variation. Selecting suitable reference genes is a prerequisite for identifying the accurate expression pattern of these genes. We analyzed the stability of eight reference genes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), protein phosphatase 2A (PP2A), β-tubulin, Actin, TIP41-like family protein (TIP41), elongation factor 1α (EF-1α), SAND family protein (SAND) and S-adenosylmethionine synthetase (SAMS) in different flower developmental stages of four different color lines in cineraria. The results showed that SAND and Actin were the most stable genes in the blue and pink lines, while the TIP41 and Actin genes performed best in the white line; however, the PP2A and Actin genes were the least variable in the carmine line. SAND and Actin genes were the most stable gene in all samples. SAMS was the least stable gene in all samples, so it is not suitable as a reference gene. In addition, the relative expression level of Senecio cruentus Flavanone 3-hydroxylase gene (ScF3H), the upstream gene in the anthocyanin biosynthesis pathway, in the different developmental stages of the flower in the carmine line was analyzed to verify the reference genes selected in this study. We recommended the combination of SAND, Actin and PP2A were suitable for normalization the target genes in different color lines of cineraria during their flower developmental stages. This study presented the first systematic study of reference genes stability and provided a guide to research of the molecular mechanism of the anthocyanin biosynthesis pathway and flower development in cineraria.

The plant-specific NAC (NAM, ATAF, and CUC) transcription factor family plays a vital role in various plant growth and developmental processes as well as in stress resistance. Using RNA sequencing, we found that the ClNAC genes (ClNAC1-44) were the most strongly up-regulated transcription factor family in Chrysanthemum lavandulifolium leaves under salt treatment. We carried out reverse transcriptase polymerase chain reaction to monitor ClNAC genes response against multiple stresses and hormonal treatments including salt, drought, cold, heat, abscisic acid and salicylic acid treatments. The results showed that 35 ClNAC genes were differentially expressed in different organ, and 32 ClNAC genes could respond to at least 2 kinds of treatments. Quantitative real time polymerase chain reaction showed that 10 ClNAC genes belonging to 7 different subfamilies could respond to at least 5 kinds of treatments. Over 50-fold variation in transcriptional levels of ClNAC17 and ClNAC21 genes was observed under 6 different types of treatments. In the present study, high-level expression of ClNAC genes under abiotic stresses and hormonal treatments suggests that the NAC transcription factors play important roles in abiotic stress tolerance and adaptation.