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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.

Chrysanthemum lavandulifolium (Fischer ex Trautv.) Makino is a diploid plant belonging to the Asteraceae family, with typical capitula composed of female ray florets and bisexual disc florets. The differentiation and development of these two types of florets have long been important research focuses; however, the potential epigenetic mechanisms governing these processes have not been elucidated. In the present study, methylation-sensitive amplification polymorphism method was used to trace the dynamic changes of DNA methylation during capitulum development in C. lavandulifolium. DNA methylation patterns and levels were detected in the whole capitula during seven developmental stages, and the obtained results revealed that DNA demethylation was dominant during this process. In addition, DNA methylation patterns and levels showed significant differences between ray and disc florets. Moreover, the expression patterns of candidate genes potentially involved in the development processes of two types of florets were analyzed by real-time quantitative reverse transcription polymerase chain reaction, and correlation analysis indicated that the expression levels of ClPI, ClAG2, ClSEP1, ClCYC2c, ClCYC2d, and ClCYC2e were highly correlated with DNA methylation levels. These results indicate that DNA methylation may be involved in the differentiation and development of ray and disc florets. This study provides epigenetic insights into the capitulum development in C. lavandulifolium.

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.

Carotenoids are the most important pigments determining the color of C. × morifolium; however, it is still unknown whether the changes of plastid ultrastructure affect carotenoids accumulation. In this study, we compared the change of carotenoid composition, content, and the plastid ultrastructures in the different developmental stages of capitulum among fourteen C. × morifolium cultivars from seven color groups. We found that the carotenoids and plastids detected at the early stage of capitulum development were similar in all cultivars, including violaxanthin, lutein, and β-carotene, which were present in proplastids and immature chloroplasts. Immature chloroplasts were degraded completely, forming loosely broken plastids during the development of the capitulum in white and pink cultivars. Meanwhile, a number of lipid vesicles appeared at proplastids, which resulted in only trace amounts of carotenoid accumulation in these cultivars. For yellow, orange, red, and brown cultivars, a great number of chromoplasts were found, which contained diverse ultrastructures, such as plastoglobules, tubules, and lipid droplets; these chromoplasts were derived from proplastids or chloroplasts. Compared with the early stage of capitulum development, these cultivars accumulated large amounts of carotenoids, primarily including lutein, lutein derivatives, and their isomers. In green cultivars, proplastids and immature chloroplasts were completely transformed into mature chloroplasts. These chloroplasts mainly contained violaxanthin, lutein, β-carotene, and two new components, (9Z)-lutein and (9'Z)-lutein, compared with carotenoid components presented in proplastids and immature chloroplasts. This research will be helpful for understanding the mechanisms of carotenoid metabolism of C. × morifolium. Furthermore, we found that two different chromoplast transformation patterns could be present in the same tissue cell, which contributed to the research on plastid differentiation and development in higher plants.

Plant growth and development are significantly affected by salt stress. Chrysanthemum lavandulifolium is a halophyte species and one of the ancestors of chrysanthemum (C. ×morifolium). Understanding how this species tolerates salt stress could provide vital insight for clarifying the salt response systems of higher plants, and chrysanthemum-breeding programs could be improved. In this study, salt tolerance was compared among C. lavandulifolium and three chrysanthemum cultivars by physiological experiments, among which C. lavandulifolium and Jinba displayed better tolerance to salt stress than the other two cultivars, whereas Xueshan was a salt-sensitive cultivar. Using the transcriptome database of C. lavandulifolium as a reference, we used digital gene expression technology to analyze the global gene expression changes in C. lavandulifolium seedlings treated with 200 mm NaCl for 12 hours compared with seedlings cultured in normal conditions. In total, 2254 differentially expressed genes (DEGs), including 1418 up-regulated and 836 down-regulated genes, were identified. These DEGs were significantly enriched in 35 gene ontology terms and 29 Kyoto Encyclopedia of Genes and Genomes pathways. Genes related to signal transduction, ion transport, proline biosynthesis, reactive oxygen species scavenging systems, and flavonoid biosynthesis pathways were relevant to the salt tolerance of C. lavandulifolium. Furthermore, comparative gene expression analysis was conducted using reverse transcription polymerase chain reaction to compare the transcriptional levels of significantly up-regulated DEGs in C. lavandulifolium and the salt-sensitive cultivar Xueshan, and species-specific differences were observed. The analysis of one of the DEGs, ClAKT, an important K+ transport gene, was found to enable transgenic Arabidopsis thaliana to absorb K+ and efflux Na+ under salt stress and to absorb K+ under drought stress. The present study investigated potential genes and pathways involved in salt tolerance in C. lavandulifolium and provided a hereditary resource for the confinement of genes and pathways responsible for salt tolerance in this species. This study provided a valuable source of reference genes for chrysanthemum cultivar transgenesis breeding.

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.

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.

Background The ray floret shapes referred to as petal types on the chrysanthemum (Chrysanthemum × morifolium Ramat.) capitulum is extremely abundant, which is one of the most important ornamental traits of chrysanthemum. However, the regulatory mechanisms of different ray floret shapes are still unknown. C. vestitum is a major origin species of cultivated chrysanthemum and has flat, spoon, and tubular type of ray florets which are the three basic petal types of chrysanthemum. Therefore, it is an ideal model material for studying ray floret morphogenesis in chrysanthemum. Here, using morphological, gene expression and transcriptomic analyses of different ray floret types of C. vestitum, we explored the developmental processes and underlying regulatory networks of ray florets. Results The formation of the flat type was due to stagnation of its dorsal petal primordium, while the petal primordium of the tubular type had an intact ring shape. Morphological differences between the two ray floret types occurred during the initial stage with vigorous cell division. Analysis of genes related to flower development showed that CYCLOIDEA genes, including CYC2b, CYC2d, CYC2e, and CYC2f, were differentially expressed in different ray floret types, while the transcriptional levels of others, such as MADS-box genes, were not significantly different. Hormone-related genes, including SMALL AUXIN UPREGULATED RNA (SAUR), GRETCHEN HAGEN3 (GH3), GIBBERELLIN 2-BETA-DIOXYGENASE 1 (GA2OX1) and APETALA2/ETHYLENE RESPONSIVE FACTOR (AP2/ERF), were identified from 1532 differentially expressed genes (DEGs) in pairwise comparisons among the flat, spoon, and tubular types, with significantly higher expression in the tubular type than that in the flat type and potential involvement in the morphogenesis of different ray floret types. Conclusions Our findings, together with the gene interactional relationships reported for Arabidopsis thaliana, suggest that hormone-related genes are highly expressed in the tubular type, promoting petal cell division and leading to the formation of a complete ring of the petal primordium. These results provide novel insights into the morphological variation of ray floret of chrysanthemum.

Flower type is an important and extremely complicated trait of chrysanthemum. The corolla tube merged degree (CTMD) and the relative number of ray florets (RNRF) are the two key factors affecting chrysanthemum flower type. However, few reports have clarified the inheritance of these two complex traits, which limits directed breeding for flower-type improvement. In this study, 305 F1hybrids were obtained from two parents with obvious differences in CTMD and RNRF performance. Using specific-locus amplified fragment sequencing (SLAF-seq) technology, we constructed a high-density genetic linkage map with an average map distance of 0.76 cM. Three major QTLs controlling CTMD and four major QTLs underlying RNRF were repeatedly detected in the 2 years. Moreover, the synteny between the genetic map and other Compositae species was investigated, and weak collinearity was observed. In QTL regions with a high degree of genomic collinearity, eight annotated genes were probed in theHelianthus annuusL. andLactuca sativaL. var. ramosa Hort. genomes. Furthermore, 20 and 11 unigenes were identified via BLAST searches between the SNP markers of the QTL regions and theC. vestitumandC. lavandulifoliumtranscriptomes, respectively. These results lay a foundation for molecular marker-assisted breeding and candidate gene exploration in chrysanthemum without a reference assembly.

Leaf shape is an important quality trait of agronomic crops, and to control the law of genetic variation of leaf shape is of practical significance for improving the early identification and selection of agronomic crops. Variations in the leaf morphology of chrysanthemum cultivars are abundant, and previous studies have quantitatively defined and classified the leaf morphology of chrysanthemum, however, the genetic architecture of chrysanthemum leaves has not been elucidated to date. In this study, two pairs of F1 hybrid populations were constructed by using small-flower chrysanthemum varieties with differences in leaf traits, and the genetic variation rules of these important quantitative traits were further discussed based on the major gene and polygene mixed inheritance analyses. The results showed that the leaves in blade shape (LBS), leaf length/width is controlled by two pairs of additive-dominant major genes (B-1), the widest part length/leaf length is controlled by two completely dominant genes (B-5), in leaf lobe shape (LLS), the lobe length/vein length is controlled by one pair of additive dominant major genes (A-1), and the lobe length/lobe width is controlled by two pairs of additive dominant major genes (B-2). The heritability of major genes was greater than 30%. For the leaf petiole shape (LPS), the petiole length is controlled by a pair of additive-dominant major genes (A-1). The results showed that the leaf traits were mainly controlled by genetic factors. In addition, based on the high-density genetic map of one F1 hybrid population, it was found that 51 quantitative trait loci (QTL) were used to control the leaf traits, including two QTLs that controlled the LBS. There were 18 QTLs that controlled LLS. Moreover, the primary QTLs that controlled leaf width and lobe length were obtained. The results of this study may establish a theoretical foundation for the in-depth exploration of leaf-shape-related genes in chrysanthemum and may provide a reference for future research investigating leaf-shape genetics in other agronomic crops.

Plants sense photoperiod signals to confirm the optimal flowering time. Previous studies have shown that Cryptochrome2 (CRY2) functions to promote floral transition in the long-day plant (LDP) Arabidopsis; however, the function and molecular mechanism by which CRY2 regulates floral transition in short-day plants (SDPs) is still unclear. In this study, we identified a CRY2 homologous gene, ClCRY2, from Chrysanthemum lavandulifolium, a typical SDP. The morphological changes in the C. lavandulifolium shoot apex and ClFTs expression analysis under SD conditions showed that adult C. lavandulifolium completed the developmental transition from vegetative growth to reproductive growth after eight SDs. Meanwhile, ClCRY2 mRNA exhibited an increasing trend from 0 to 8 d of SD treatment. ClCRY2 overexpression in wild-type (WT) Arabidopsis and C. lavandulifolium resulted in early flowering. The transcript levels of the CONSTANS-like (COL) genes ClCOL1, ClCOL4, and ClCOL5, and FLOWERING LOCUS T (FT) homologous gene ClFT1 were upregulated in ClCRY2 overexpression (ClCRY2-OE) C. lavandulifolium under SD conditions. The transcript levels of some circadian clock-related genes, including PSEUDO-REPONSE REGULATOR 5 (PRR5), ZEITLUPE (ZTL), FLAVIN-BINDING KELCH REPEAT F-BOX 1 (FKF1), and GIGANTEA (GI-1 and GI-2), were upregulated in ClCRY2-OE C. lavandulifolium, while the expression levels of other circadian clock-related genes, such as EARLY FLOWERING 3 (ELF3), ELF4, LATE ELONGATED HYPOCOTYL (LHY), PRR73, and REVEILLE8 (RVE8), were downregulated in ClCRY2-OE C. lavandulifolium under SD conditions. Taken together, the results suggest that ClCRY2 promotes floral transition by fine-tuning the expression of circadian clock-related gene, ClCOLs and ClFT1 in C. lavandulifolium under SD conditions., Genetics: Harnessing flower power in chrysanthemums Researchers have identified the genes that control the timing of flowering in chrysanthemums. For most plants, day-length indicates that it is time to flower. Greenhouse growers can produce out-of-season flowers by manipulating day length, but the process is expensive. Breeding varieties that respond more quickly could reduce costs, but requires a better understanding of the underlying genetics, especially in the subset of plants triggered by short days. Si-lan Dai and co-workers at Beijing Forestry University in China investigated which genes trigger flowering in Chrysanthemum lavandulifolium, a typical short-day plant. They found that flowering of chrysanthemum is controlled by a gene previously identified in long-day plants, and that amplifying the gene produced plants that flowered much sooner than ordinary plants. These findings may help in breeding varieties that are easily induced to bloom.

The generation of chrysanthemum (Chrysanthemum &times, morifolium) flower color is mainly attributed to the accumulation of anthocyanins. Light is one of the key environmental factors that affect the anthocyanin biosynthesis, but the deep molecular mechanism remains elusive. In our previous study, a series of light-induced structural and regulatory genes involved in the anthocyanin biosynthetic pathway in the chrysanthemum were identified using RNA sequencing. In the present study, differentially expressed proteins that are in response to light with the capitulum development of the chrysanthemum &lsquo, Purple Reagan&rsquo, were further identified using isobaric tags for relative and absolute quantification (iTRAQ) technique, and correlation between the proteomic and the transcriptomic libraries was analyzed. In general, 5106 raw proteins were assembled based on six proteomic libraries (three capitulum developmental stages &times, two light treatments). As many as 160 proteins were differentially expressed between the light and the dark libraries with 45 upregulated and 115 downregulated proteins in response to shading. Comparative analysis between the pathway enrichment and the gene expression patterns indicated that most of the proteins involved in the anthocyanin biosynthetic pathway were downregulated after shading, which was consistent with the expression patterns of corresponding encoding genes, while five light-harvesting chlorophyll a/b-binding proteins were initially downregulated after shading, and their expressions were enhanced with the capitulum development thereafter. As revealed by correlation analysis between the proteomic and the transcriptomic libraries, GDSL esterase APG might also play an important role in light signal transduction. Finally, a putative mechanism of light-induced anthocyanin biosynthesis in the chrysanthemum was proposed. This study will help us to clearly identify light-induced proteins associated with flower color in the chrysanthemum and to enrich the complex mechanism of anthocyanin biosynthesis for use in cultivar breeding.

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.

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.

Cymbidium longibracteatum is a common cultivated species in the genus Cymbidium due to its elegant appearance, rich flower colors and strong fragrance, but its classification is quite controversial. In this study, the complete chloroplast genome of C. longibracteatum was obtained by Illumina sequencing. The chloroplast genome of C. longibracteatum is 150,070 bp in length with an overall GC content of 37.12%, which contains a large single-copy (LSC;84,949 bp) region, a small single-copy (SSC;13,745bp) region, and a pair of inverted repeats (IRs; 25,688 bp) regions. The genome contains 130 genes, namely 84 protein-coding genes, 38 tRNA genes and 8 rRNA genes. The maximum-likelihood phylogenetic tree has proved that C. longibracteatum should exist as an independent species in the genus Cymbidium, and it is most closely related to C. tortisepalum. This study provides valuable sequence resources for further study of C. longibracteatum.

The generation of chrysanthemum (Chrysanthemum &times, morifolium) flower color is mainly attributed to the accumulation of anthocyanins. In the anthocyanin biosynthetic pathway in chrysanthemum, although all of the structural genes have been cloned, the regulatory function of R2R3-MYB transcription factor (TF) genes, which play a crucial role in determining anthocyanin accumulation in many ornamental crops, still remains unclear. In our previous study, four light-induced R2R3-MYB TF genes in chrysanthemum were identified using transcriptomic sequencing. In the present study, we further investigated the regulatory functions of these genes via phylogenetic and alignment analyses of amino acid sequences, which were subsequently verified by phenotypic, pigmental, and structural gene expression analyses in transgenic tobacco lines. As revealed by phylogenetic and alignment analyses, CmMYB4 and CmMYB5 were phenylpropanoid and flavonoid repressor R2R3-MYB genes, respectively, while CmMYB6 was an activator of anthocyanin biosynthesis, and CmMYB7 was involved in regulating flavonol biosynthesis. Compared with wild-type plants, the relative anthocyanin contents in the 35S:CmMYB4 and 35S:CmMYB5 tobacco lines significantly decreased (p &lt, 0.05), while for 35S:CmMYB6 and 35S:CmMYB7, the opposite result was obtained. Both in the 35S:CmMYB4 and 35S:CmMYB5 lines, the relative expression of several anthocyanin biosynthetic genes in tobacco was significantly downregulated (p &lt, 0.05), on the contrary, several genes were upregulated in the 35S:CmMYB6 and 35S:CmMYB7 lines. These results indicate that CmMYB4 and CmMYB5 negatively regulate anthocyanin biosynthesis in chrysanthemum, while CmMYB6 and CmMYB7 play a positive role, which will aid in understanding the complex mechanism regulating floral pigmentation in chrysanthemum and the functional divergence of the R2R3-MYB gene family in higher plants.

Centaurea cyanus is a popular garden plant native to Europe. Although their petals show abundant colour variations, the flavonoid profiling and the potential molecular mechanisms remain unclear. In the present study, we collected six cornflower cultivars with white, pink, red, blue, mauve and black petals. Ultra-performance liquid chromatography coupled with photodiode array and tandem mass spectrometry (UPLC-MS/MS) was used to investigate the comparative profiling of flavonoids both qualitatively and quantitatively. Ten anthocyanins, six flavones and two flavonols were separated and putatively identified. Except for white petals without any anthocyanins, both pink and red flowers contained pelargonidin derivatives, whereas blue, mauve and black petals accumulated cyanidins. The expression patterns of genes involved in the flavonoid biosynthesis were performed by real-time quantitative reverse transcription-PCR. The anthocyanin biosynthetic pathway in white petals was inhibited starting from flavanone 3-hydroxylase, resulting in the absence of anthocyanin accumulation. The open reading frame of flavonoid 3'-hydroxylase in pink and red petals was truncated; this led to loss of a haem binding site, a conserved motif in the cytochrome P450 family, and loss of conversion from dihydrokaempferol to dihydroquercetin. The significantly higher expression of structural genes corresponding to the hyper-accumulation of flavonoids in black petals may play an important role in black coloration. Remarkably, the mauve and blue petals accumulated the same cyanidin derivative but contained apigenin with different modifications on the 4' position, which may cause the coloration differences. The results obtained in this study will provide insights into the mechanisms of vivid colour diversities in cornflower.

Background Roses are important plants for human beings with pivotal economical and biological traits like continuous flowering, flower architecture, color and scent. Due to frequent hybridization and high genome heterozygosity, classification of roses and their relatives remains a big challenge. Results Here, to identify potential markers for phylogenetic reconstruction and to reveal the patterns of natural selection in roses, we generated sets of high quality and comprehensive reference transcriptomes for Rosa chinensis ‘Old Blush’ (OB) and R. wichuriana ‘Basye’s Thornless’ (BT), two species exhibiting contrasted traits of high economical importance. The assembled reference transcriptomes showed transcripts N50 above 2000 bp. Two roses shared about 10,073 transcripts (N50 = 2282 bp), in which a set of 5959 transcripts was conserved within genera of Rosa. Further comparison with species in Rosaceae identified 4447 transcripts being common (Rosaceae-common) in Rosa, Malus, Prunus, Rubus, and Fragaria, while a pool of 164 transcripts being specific for roses (Rosa-specific). Among the Rosaceae-common transcripts, 409 transcripts showed a signature of positive selection and a clustered expression in different tissues. Interestingly, nine of these rapidly evolving genes were related to DNA damage repair and responses to environmental stimulus, a potential associated with genome confliction post hybridization. Coincident with this fast evolution pattern in rose genes, 24 F-box and four TMV resistant proteins were significantly enriched in the Rosa-specific genes. Conclusions We expect that these Rosaceae-common and Rosa-specific transcripts should facilitate the phylogenetic analysis of Rosaceae plants as well as investigations of Rosa-specific biology. The data reported here could provide fundamental genomic tools and knowledge critical for understanding the biology and domestication of roses and for roses breeding. Electronic supplementary material The online version of this article (10.1186/s12870-018-1585-x) contains supplementary material, which is available to authorized users.

The light-dependent coloration of the vital organs of horticultural crops affects multiple parts of production and sales. The simplicity of the metabolic pathways of anthocyanins and the characteristics of light-dependent coloration make chrysanthemum (Chrysanthemum ×morifolium) an ideal subject for studying the mechanism of light-regulated anthocyanin biosynthesis. In this study, real-time quantitative reverse transcription–polymerase chain reaction (PCR) was used in the analysis of the expression levels of anthocyanin biosynthesis genes in C. ×morifolium ‘Reagan’. The reference genes selected were those assumed to remain at constant levels in three flower color lines at five floral developmental stages and at two light conditions. Using digital gene expression technology, we selected nine reference genes with moderate expression in the chrysanthemum ray florets at various floral developmental stages under illuminated and dark conditions as the candidate reference genes for further study. After comprehensively analyzing the stability of gene expression with three distinct statistical algorithms, geNorm, NormFinder, and qBase plus, we found that F-box and PP2A were the most stable genes in all of the samples. In addition, we analyzed the relative expression level of the CmF3H gene in different samples to verify the reference genes that we selected. This study provides a consensus list of validated reference genes that will benefit future studies of the expression of chrysanthemum genes involved in anthocyanin biosynthesis and floral development under various light conditions. Moreover, this information will also promote the molecular breeding of horticultural crops for their color modification.

This paper reports the first study on plant CBF transcription factors (TF) in salt and drought stress responses in Chrysanthemum lavandulifolium. A CBF homolog gene, named as ClCBF1, from C. lavandulifolium was isolated using rapid amplification of cDNA ends (RACE). The deduced peptide is comprised of 210 amino acids (AA) containing an AP2 structural domain characteristic of the AP2 gene family. Quantitative real-time PCR revealed that ClCBF1 gene exhibit differential expression patterns across root, leaf and stem tissues, and it was strongly induced under salt and drought treatments of C. lavandulifolium. Overexpression of ClCBF1 in C. morifolium ‘White Snow’ resulted in stronger tolerance to salt and drought stresses. The ClCBF1 expression level, enzymatic activities of superoxide dismutase and peroxidase, and contents of proline and soluble proteins were enhanced in these transgenic lines, they were repressed in the antisense transgenic lines under the same stress conditions. Results indicate that ClCBF1 represents a promising candidate gene in improving abiotic stress tolerance among ornamental plants.

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.

The MADS-box gene SOC1/TM3 (SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1/ Tomato MADS-box gene 3) is a main integrator in the Arabidopsis flowering pathway; its structure and function are highly conserved in many plant species. SOC1-like genes have been isolated in chrysanthemum, one of the most well-known ornamental plants, but it has not been well characterized thus far. We isolated and characterized ClSOC1-1 and ClSOC1-2, two putative orthologs of Arabidopsis SOC1, from the wild diploid chrysanthemum, Chrysanthemum lavandulifolium, to investigate the regulatory mechanisms of flowering time control in chrysanthemum. Expression analysis indicated that ClSOC1-1 and ClSOC1-2 were expressed in all examined organs/tissues (leaves, shoot apices, petioles, stems and roots) with different expression levels, and with high expression in the shoot apices and leaves during the early stage of floral transition. The expression levels of ClSOC1-1 and ClSOC1-2 in the shoot apices increased at different developmental stages with the highest expression levels after 7 days of short-day treatment. Overexpression of ClSOC1-1 and ClSOC1-2 in wild-type Arabidopsis resulted in early flowering, which was coupled with the upregulation of one of the flowering promoter genes LEAFY. Our results suggested that the ClSOC1-1 and ClSOC1-2 genes play an evolutionarily conserved role in promoting flowering in Chrysanthemum lavandulifolium and could serve as a vital target for the genetic manipulation of flowering time in the chrysanthemum.

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 cyanidin (Cy), pelargonidin (Pg), and delphinidin (Dp) pathways are the three major branching anthocyanin biosynthesis pathways that regulate flavonoid metabolic flux and are responsible for red, orange, and blue flower colors, respectively. Different species have evolved to develop multiple regulation mechanisms that form the branched pathways. In the current study, five Senecio cruentus cultivars with different colors were investigated. We found that the white and yellow cultivars do not accumulate anthocyanin and that the blue, pink, and carmine cultivars mainly accumulate Dp, Pg, and Cy in differing densities. Subsequent transcriptome analysis determined that there were 43 unigenes encoding anthocyanin biosynthesis genes in the blue cultivar. We also combined chemical and transcriptomic analyses to investigate the major metabolic pathways that are related to the observed differences in flower pigmentation in the series of S. cruentus. The results showed that mutations of the ScbHLH17 and ScCHI1/2 coding regions abolish anthocyanin formation in the white and the yellow cultivars; the competition of the ScF3'H1, ScF3'5'H, and ScDFR1/2 genes for naringenin determines the differences in branching metabolic flux of the Cy, Dp, and Pg pathways. Our findings provide new insights into the regulation of anthocyanin branching and also supplement gene resources (including ScF3'5 'H, ScF3'H, and ScDFRs) for flower color modification of ornamentals.

Quantitative real-time PCR (qPCR) is a popular and powerful tool used to understand the molecular mechanisms of flower development. However, the accuracy of this approach depends on the stability of reference genes. The capitulum of chrysanthemums is very special, which is consisting of ray florets and disc florets. There are obvious differences between the two types of florets in symmetry, gender, histological structure, and function. Furthermore, the ray florets have various shapes. The objective of present study was to identify the stable reference genes in Chrysanthemum morifolium and Chrysanthemum lavandulifolium during the process of flower development. In this study, nine candidate reference genes were selected and evaluated for their expression stability acrosssamples during the process of flower development, and their stability was validated by four different algorithms (Bestkeeper, NormFinder, GeNorm, and Ref-finder). SAND (SAND family protein) was found to be the most stably expressed gene in all samples or different tissues during the process of C. lavandulifolium development. Both SAND and PGK (phosphoglycerate kinase) performed most stable in Chinese large-flowered chrysanthemum cultivars, and PGK was the best in potted chrysanthemums. There were differences in best reference genes among varieties as the genetic background of them were complex. These studies provide guidance for selecting reference genes for analyzing the expression pattern of floral development genes in chrysanthemums.

A subtractive cDNA library was developed to study genes associated with the release of dormant buds in tree peony. To identify genes that are highly expressed in buds released from dormancy, 588 clones were examined by differential screening and then 185 clones were selected to be sequenced. A total of 31 unique genes were obtained, of which only 25 sequences had matches in the NCBI database or Arabidopsis thaliana protein database while 6 sequences with no matches. Many of the different genes were identified as having unknown or hypothetical functions while others were speculated to have different molecular functions. The expression profiles of the selected “candidate” genes which may be associated with dormancy release according to their putative function and previous reports were assessed by northern blot and semi-quantitative RT-PCR. The results indicated that the transcriptional expressions of the isolated genes are related to growth regulation and stress response. Our results provide interesting information for further understanding the molecular mechanism of bud dormancy release in tree peony.

A subtractive cDNA library was developed to study genes associated with bud dormancy release in tree peonies. In order to identify genes that are highly expressed in buds released from dormancy, 588 clones were examined by differential screening. Of these, 185 clones were selected to be sequenced. A total of 37 unique sequences were obtained of which only 31 sequences have matches in the NCBI database or the Arabidopsis thaliana protein database. Semi-quantitative RT-PCR was used to confirm further the expression profiles for 12 transcripts identified within the subtractive cDNA library. Gene ontology analyses indicated that many of the different genes identified have unknown or hypothetical functions while it is speculated that other genes play different molecular roles. In our study, genes involved in bud dormancy release were growth-related or stress-responsive, while low-temperature-induced ribosomal proteins may also play a role in bud dormancy release. Our results provide interesting information for further understanding of the molecular mechanism of bud dormancy release in tree peonies.

Background The flower colour of agricultural products is very important for their commercial value, which is mainly attributed to the accumulation of anthocyanins. Light is one of the key environmental factors that affect the anthocyanin biosynthesis. However, the deep molecular mechanism remains elusive, and many problems regarding the phenotypic change and the corresponding gene regulation are still unclear. In the present study, Chrysanthemum × morifolium ‘Purple Reagan’, a light-responding pigmentation cultivar, was selected to investigate the mechanism of light-induced anthocyanin biosynthesis using transcriptomic analyses. Results Only cyanidin derivatives were identified based on the analyses of the pigmentation in ray florets. Shading experiments revealed that the capitulum was the key organ and that its bud stage was the key phase responding to light. These results were used to design five libraries for transcriptomic analyses, including three capitulum developmental stages and two light conditions. RNA sequences were de novo assembled into 103,517 unigenes, of which 60,712 were annotated against four public protein databases. As many as 2,135 unigenes were differentially expressed between the light and dark libraries with 923 up-regulated and 1,212 down-regulated unigenes in response to shading. Next, interactive pathway analysis showed that the anthocyanin biosynthetic pathway was the only complete metabolic pathway both modulated in response to light and related to capitulum development. Following the shading treatment, nearly all structural genes involved in the anthocyanin biosynthetic pathway were down-regulated. Moreover, three CmMYB genes and one CmbHLH gene were identified as key transcription factors that might participate in the regulation of anthocyanin biosynthesis under light conditions based on clustering analysis and validation by RT-qPCR. Finally, a light-induced anthocyanin biosynthesis pathway in chrysanthemums was inferred. Conclusion The pigmentation of the ray florets of chrysanthemum cultivar ‘Purple Reagan’ is dependent on light. During the light-induced pigmentation process, the expression of seven structural genes in the anthocyanin biosynthetic pathway (regulated by at least four transcription factors in response to light) are the main contributors to the pigmentation of chrysanthemums. This information will further our understanding of the molecular mechanisms governing light-induced anthocyanin biosynthesis in ornamental plants. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1428-1) contains supplementary material, which is available to authorized users.

The CONSTANS (CO) and CONSTANS-like (COL) genes play key roles in the photoperiodic flowering pathways, and studying their functions can elucidate the molecular mechanisms underlying flowering control in photoperiod-regulated plants. We identified eleven COL genes (ClCOL1–ClCOL11) in Chrysanthemum lavandulifolium and divided them into three groups that are conserved among the flowering plants based on phylogenetic analysis. Most of the ClCOL genes are primarily expressed in the leaf and shoot apices, except for ClCOL6–ClCOL9, which belong to Group II. The expression levels of ClCOL4–ClCOL5 and ClCOL7–ClCOL8 are up-regulated under inductive short-day (SD) conditions, whereas ClCOL6 is down-regulated under inductive SD conditions. The ClCOL genes exhibit four different diurnal rhythm expressions (Type I–Type IV). The Type I genes (ClCOL4–ClCOL5) are highly transcribed under light. The Type II genes (ClCOL1–ClCOL2, ClCOL10) display increased expression in darkness and are rapidly suppressed under light. Transcripts of ClCOL6–ClCOL9 and ClCOL11, belonging to Type III, are abundant in the late light period or at the beginning of the dark period. ClCOL3 belongs to Type IV, with high expression in the early light period and dark period. The peak expression levels of ClCOL4–ClCOL6 are decreased and postponed in the non-inductive night break (NB) and under long-day (LD) conditions, indicating that those genes may play an essential role in the flowering regulation of C. lavandulifolium. The overexpression of ClCOL5 promotes the flowering of Arabidopsis grown under LD conditions, suggesting that ClCOL5 may function as a flowering enhancer in C. lavandulifolium. This study will be useful not only for the study of the C. lavandulifolium photoperiod-dependent flowering process but also for the genetic manipulation of flowering time-related genes to change the flowering time in the chrysanthemum.

The FLOWERING LOCUS T (FT) gene plays crucial roles in regulating the transition from the vegetative phase to the reproductive phase. In this study, we isolated an FT homologous gene (denoted as ClFT) from Chrysanthemum lavandulifolium. The sequencing analysis indicated that the promoter of the ClFT gene contains many elements, such as light response, abscisic acid, drought-inducibility response and CIRCADIAN clock elements. The expression patterns of ClFT in different tissues/organs at different developmental stages and its responses to different photoperiods were observed. ClFT is expressed in all tested organs/tissues, with the highest expression level being observed in the leaves of plants with visible floral buds under the short day (SD) condition. Next, we studied the rhythmic expression of ClFT during different photoperiod treatments and found that the level of ClFT increases with additional hours of continuous dark. ClFT accumulates when the continuous dark period is 12 h, regardless of the duration of light period. The ectopic expression of the ClFT gene in wild type Arabidopsis (Col-0) results in early flowering, with high expression levels of endogenous LFY and SOC1 being observed in transgenic Arabidopsis. All results indicated that the ClFT gene plays an evolutionarily conserved role in promoting flowering in inductive short days in C. lavandulifolium and that this gene could serve as a vital target for the genetic manipulation of flowering time in chrysanthemums.

As the key enzyme in the biosynthesis of blue flower color pigments, flavonoid 3',5'-hydroxylase (F3'5'H) can catalyze the conversion of its major substrates, 2-S naringenin and dihydrokaempferol, into 3',4',5'-hydroxylated pentahydroxyflavanone and dihydromyricetin, respectively. Unlike other F3'5'Hs belonging to the CYP75A subfamily, Asteraceae-specific F3'5'Hs belong to the CYP75B subfamily. Furthermore, cineraria F3'5'H expressed in yeast exhibited not only F3'H (flavonoid 3'-hydroxylase) activity but also F3'5'H activity in vitro. In this study, Southern blotting showed that there was only one copy of a homolog of the F3'5'H gene PCFH in the Pericallis × hybrida genome. This gene could be detected by Northern blot in the primary developmental stages of ligulate florets of the purple- and blue-flowered cultivars, and its transcripts also accumulated in the leaves. Heterologous expression of PCFH could produce new delphinidin derivatives in the corollas of transgenic tobacco plants, increased the content of cyanidin derivatives and lead to the blue- and red-shifting of flower color in T₀ generation plants. These results indicate that cineraria F3'5'H exhibited both F3'5'H- and F3'H-activity in vivo. The types and contents of anthocyanins and flower color phenotypes of the T₁ generation were similar to those of T₀ generation plants. PCFH exhibited stable inheritance and normal functions between generations. This study supplies new evidence to understand Asteraceae-specific F3'5'Hs and provides important references for the further study of molecular breeding of blue-flowered chrysanthemums using the PCFH gene.

To investigate the diversity of root endophytes in Rhododendron fortunei, fungal strains were isolated from the hair roots of plants from four habitats in subtropical forests of China. In total, 220 slow-growing fungal isolates were isolated from the hair roots of R. fortunei. The isolates were initially grouped into 17 types based on the results of internal transcribed spacer-restriction fragment length polymorphism (ITS-RFLP) analysis. ITS sequences were obtained for representative isolates from each RFLP type and compared phylogenetically with known sequences of ericoid mycorrhizal endophytes and selected ascomycetes or basidiomycetes. Based on phylogenetic analysis of the ITS sequences in GenBank, 15 RFLP types were confirmed as ascomycetes, and two as basidiomycetes; nine of these were shown to be ericoid mycorrhizal endophytes in experimental cultures. The only common endophytes of R. fortunei were identified as Oidiodendron maius at four sites, although the isolation frequency (3-65%) differed sharply according to habitat. Phialocephala fortinii strains were isolated most abundantly from two habitats which related to the more acidic soil and pine mixed forests. A number of less common mycorrhizal RFLP types were isolated from R. fortunei at three, two, or one of the sites. Most of these appeared to have strong affinities for some unidentified root endophytes from Ericaceae hosts in Australian forests. We concluded that the endophyte population isolated from R. fortunei is composed mainly of ascomycete, as well as a few basidiomycete strains. In addition, one basidiomycete strain was confirmed as a putative ericoid mycorrhizal fungus.

A cDNA clone was isolated from tree peony (Paeonia suffruticosa) subtractive cDNA library of burst buds and characterized with regard to its sequence, expression in response to chilling treatment during the release of bud dormancy, and its function in transgenic Arabidopsis thaliana. The clone, designated as PsMPT, contains 1,615 nucleotides with an open reading frame of 1,119 nucleotides, and the deduced amino acid sequence shows high homology with mitochondrial phosphate transporters (MPTs) from various organisms. The mRNA accumulation of PsMPT in tree peony was strongly induced by chilling treatment during the release of bud dormancy. When the treated plants were transferred to normal growth conditions, the level of PsMPT transcripts induced by sufficient chilling could be maintained high, whereas that induced by insufficient chilling decreased sharply. The transgenic Arabidopsis plants that overexpress PsMPT showed rapid growth and earlier flowering than wild-type plants. ATP contents in the transgenic plants were much higher than that in wild-type plants through various developmental stages. Together, these results suggest that the product of PsMPT is a MPT and might play an important role during the release of bud dormancy in tree peony.