Isolation and functional analysis of a homolog of flavonoid 3',5'-hydroxylase gene from Pericallis × hybrida.

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.

ClCRY2 facilitates floral transition in Chrysanthemum lavandulifolium by affecting the transcription of circadian clock-related genes under short-day photoperiods.

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.

Comparative analyses of light-induced anthocyanin accumulation and gene expression between the ray florets and leaves in chrysanthemum.

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.

DFL, a FLORICAULA/LEAFY homologue gene from Dendranthema lavandulifolium is expressed both in the vegetative and reproductive tissues.

FLO/LFY homologue genes were initially characterized as floral meristem identity genes and play a key role in flower development among diverse species. The inflorescence organization of chrysanthemum differs from typical dicotyledons such as Arabidopsis and Antirrhinum as clear sepals are absent, and instead, a pappus, a rudimentary sepal, is formed. To understand the mechanism of reproduction of chrysanthemum at the molecular level, DFL, a FLORICAULA/LEAFY homologous gene, was cloned from Dendranthema lavandulifolium, which is one of the original species of chrysanthemum. The DFL gene consists of a 1,236-bp open reading frame and encodes a putative protein of 412 amino acids, which is 63% identical to LFY and 70% to FLO. The expression patterns of DFL during the flower development were analyzed, and RT-PCR results showed that DFL was strongly expressed in the flower bud. In situ hybridization experiments showed that it is strongly expressed in the inflorescence bract, petal and stamen primordial tissues throughout the inflorescence development. Its expression signals were also detected in stems, leaf primordial tissues and developing inflorescence bracts.