Jiaying Wang

Chrysanthemum (Chrysanthemum × morifolium Ramat.) is a globally popular ornamental plant, but most cultivars lack efficient petal-based transient transformation systems, limiting floral trait molecular mechanism exploration. Protoplasts are versatile tools for gene localization, interaction, and functional characterization. Here, we established a petal protoplast isolation and transient transformation system for C. morifolium ‘Wandai Fengguang’ via L₉(3⁴) orthogonal design: optimal isolation (0.6 M mannitol, 8 h enzymatic digestion time, 0.4% macerozyme R-10, 4% cellulase R-10) and transformation (40% PEG4000, 12 μg plasmid, 10 min transfection, a protoplast density of 1 × 10⁶ protoplasts mL⁻¹). Under these conditions, protoplast yield was 5.14 × 10⁶ protoplasts g⁻¹·FW, viability 87.41%, and transformation efficiency 51.50%, with good applicability for six additional germplasms. We further analyzed CmVIT1 protein localization. Compared with the previous system, this system significantly improved protoplast yield and transformation efficiency, facilitating the transient transformation of genes related to floral traits in chrysanthemum and providing a methodological framework for other horticultural crops.

Chrysanthemum × morifolium Ramat. is a globally renowned ornamental flower. It includes numerous varieties, most of which are typical short-day (SD) plants, and the flowering characteristics of different chrysanthemum varieties in response to the photoperiod vary greatly. In this study, seven representative chrysanthemum varieties were selected for a comparative analysis of flowering traits under long-day conditions (16 h/8 h day/night) and short-day conditions (12 h/12 h day/night). It was found that three varieties (‘A44’, ‘C60’, and ‘183’) belonged to obligatory short-day varieties and four varieties (‘A20’, ‘C1’, ‘C27’, and ‘C31’) belonged to facultative short-day varieties. The short-day conditions not only induced earlier flowering but also improved flowering quality in the facultative SD varieties. Different chrysanthemum varieties required different light conditions to complete the vegetative stage and reach the floral competent state. Seven chrysanthemum varieties, ‘A44’, ‘C60’, ‘183’, ‘A20’, ‘C1’, ‘C27’, and ‘C31’, reached a floral competent state in the L20, L20, L22, L22, L18, L20, and L24 periods, respectively, and were most sensitive to SD induction at this time. The expression patterns of key floral genes in the photoperiod pathway were analyzed and it was found that CmCRY1, CmCRY2, CmGI1, CmGI2, and CmCO were mainly expressed in leaves. Then, comparing the expression levels of these genes under LD and SD conditions, the expression of CmGI1, CmGI2, CmCO, and CmFTL were not significantly induced in the obligatory SD varieties, while the expression of them in the facultative SD varieties were induced by SD conditions. This may be the reason why the facultative varieties could respond to SD conditions more quickly to complete the floral transition. In addition, SD induction under different photoperiodic conditions and growth states resulted in differences in the phenotype of flowering. This result provides guidance for the artificial regulation of chrysanthemum flowering and improvement of ornamental quality, as well as clues for analyzing the flowering mechanism of chrysanthemums under different photoperiod conditions.

Chrysanthemum × morifolium has great ornamental and economic value on account of its exquisite capitulum. However, previous studies have mainly focused on the corolla morphology of the capitulum. Such an approach cannot explain the variable inflorescence architecture of the chrysanthemum. Previous research from our group has shown that NO APICAL MERISTEM (ClNAM) is likely to function as a hub gene in capitulum architecture in the early development stage. In the present study, ClNAM was used to investigate the function of these boundary genes in the capitulum architecture of Chrysanthemum lavandulifolium, a closely related species of C. × morifolium in the genus. Modification of ClNAM in C. lavandulifolium resulted in an advanced initiation of the floral primordium at the capitulum. As a result, the receptacle morphology was altered and the number of florets decreased. The ray floret corolla was shortened, but the disc floret was elongated. The number of capitula increased significantly, arranged in more densely compounded corymbose synflorescences. The yeast and luciferase reporter system revealed that ClAP1, ClRCD2, and ClLBD18 target and activate ClNAM. Subsequently, ClNAM targets and activates ClCUC2a/c, which regulates the initiation of floral and inflorescence in C. lavandulifolium. ClNAM was also targeted and cleaved by cla-miR164 in this process. In conclusion, this study established a boundary gene regulatory network with cla-miR164-ClNAM as the hub. This network not only influences the architecture of capitulum, but also affects compound corymbose synflorescences of the C. lavandulifolium. These results provide new insights into the mechanisms regulating inflorescence architecture in chrysanthemum.

The sepal color of Fuchsia hybrida is colorful instead of green and usually varies from the petal colors, which greatly increases its ornamental value and attract customers’ preference. However, the potential molecular mechanism underlying the color variation between sepals and petals remains unclear. The present study collected F. hybrida with red sepals and purple petals to explore the key pigments and genes involved in color development using a targeted metabolome and transcriptome. A total of 43 metabolites with diverse hydroxylation, glycosylation, methylation and acylation patterns were isolated and identified by UPLC-MS/MS. The quantification analysis showed that peonidin-3,5-O-diglucoside and malvidin-3,5-O-diglucoside were the most abundant anthocyanins accumulating in the sepals and petals, respectively. Then, six libraries from the sepals and petals were constructed for the transcriptome and 70,135 unigenes were generated. The transcript level of FhF3′H was significantly higher in the sepals, while Fh3′5′H showed more abundant expression in the petals, which can account for the abundant peonidin and malvidin accumulation in the sepals and petals, respectively. The subsequent multiomics analysis showed that both the differentially accumulated anthocyanins and expressed unigenes were enriched in the anthocyanin biosynthesis pathway. Additionally, FhMYBs potentially regulating anthocyanin biosynthesis were screened out by correlation analysis and protein interaction prediction. These findings help to elucidate the molecular mechanisms underlying the color variation between the sepals and petals in F. hybrida.

The winter pot kalanchoe (Kalanchoe blossfeldiana) is an ornamental plant with succulent leaves and clustered flowers in Crassulaceae, widely used as a potted flower or garden decoration. In nature, the bicolor petal is an interesting phenomenon, and breeders have succeeded in cultivating the winter pot kalanchoe with bicolored petals. However, its potential molecular mechanism of pigmentation is poorly understood. This study collected a yellow-red colored winter pot kalanchoe to investigate the molecular mechanism underlying its bicolor formation using the integrative analyses of metabolome and transcriptome. The metabolome results showed that both flavonoid and carotenoid co-existed in the winter pot kalanchoe petals, whereas only anthocyanin accumulation showed significant differences—about nineteen times higher in the red region than that in the yellow region. The differentially expressed genes were significantly enriched in the anthocyanin biosynthesis pathway, and the expression level of biosynthetic genes, including KbCHS, KbCHI, KbF3H, KbDFR, KbANS and KbGTs, were significantly upregulated in the red region. Moreover, transcription factors potentially regulating anthocyanin biosynthesis were predicted, and KbMYB2 and KbbHLH1 might play important roles in positively regulating anthocyanin biosynthesis in the red region. The findings reported here provide new insights into the understanding of petal bicolor formation mechanisms and will assist cultivar innovation in winter pot kalanchoe.