A group VIIIa ethylene-responsive factor, CmERF4, negatively regulates waterlogging tolerance in chrysanthemum.

Ethylene-responsive factors (ERF) play an important role in plant responses to waterlogging stress. However, the function and mechanism of action of ERFVIII in response to waterlogging stress remain poorly understood. In this study, we found that expression of the ERF VIIIa gene CmERF4 in chrysanthemum was induced by waterlogging stress. CmERF4 localized to the nucleus when expressed in tobacco leaves. Yeast two-hybrid and luciferase assays showed that CmERF4 is a transcriptional inhibitor. CmERF4 overexpression in chrysanthemum reduced plant waterlogging tolerance, whereas overexpression of the chimeric activator CmERF4-VP64 reversed its transcriptional activity, promoting higher waterlogging tolerance than that observed in wild-type plants, indicating that CmERF4 negatively regulates waterlogging tolerance. Transcriptome profiling showed that energy metabolism and reactive oxygen species (ROS) pathway-associated genes were differentially expressed between CmERF4-VP64 and wild-type plants. RT-qPCR analysis of selected energy metabolism and reactive oxygen species-related genes showed that the gene expression patterns were consistent with the expression levels obtained from RNA-seq analysis. Overall, we identified new functions of CmERF4 in negatively regulating chrysanthemum waterlogging tolerance by modulating energy metabolism and ROS pathway genes.

Morphological Characteristics and Expression Patterns of CmCYC2c of Different Flower Shapes in Chrysanthemum morifolium.

The chrysanthemum is widely used as a cut flower, potted flower, and garden flower worldwide and has high ornamental, edible, and medicinal value. The flower heads, composed of ray florets and disc florets, are the most diverse in terms of morphology among ornamental plants. Here, we compared and analyzed the developmental processes of different capitulum types as well as ray florets and disc florets. Morphological differentiation of the two florets occurred on the dorsal domain of the petals at stage Ⅳ of flower development, and differences in stamen development occurred at stage Ⅴ. The dorsal domain of the ray florets and the early stage of flower development were also an essential site and period, respectively, for the differences among capitulum types. In situ hybridization revealed that CmCYC2c, whose homologs are involved in the specification of floret identity in Asteraceae, was expressed in both the dorsal and ventral domains of the ray petals in the tubular-type chrysanthemum, whereas, it was differentially transcribed in the ray petals of flat- and spoon-type chrysanthemum cultivars and had lower or no expression in the dorsal domain and higher expression in the ventral domain at stage Ⅳ. Our study indicates that the expression pattern of CmCYC2c on the dorsal domain of the ray floret at stage Ⅳ contributes to the formation of diverse flower head types in chrysanthemums.

FUL homologous gene CmFL1 is involved in regulating flowering time and floret numbers in Chrysanthemum morifolium.

Flowering time and floret numbers are important ornamental characteristics of chrysanthemums that control their adaptability and inflorescence morphology, respectively. The FRUITFULL (FUL) gene plays a key role in inducing flowering and inflorescence meristem development. In this study, we isolated a homolog of the MADS-box gene FUL, CmFUL-Like 1 (CmFL1), from chrysanthemum inflorescence buds. Quantitative RT-PCR and in situ analyses showed that CmFL1 was strongly expressed in young inflorescence buds. Overexpression of CmFL1 caused early flowering while co-suppression expression of CmFL1 increased the number of florets. Furthermore, the floral promoting factors CmSOC1, CmFDL1, and CmLFY were up-regulated in the shoot tips of transgenic plants. In addition, RNA-seq analysis of the transgenic plants suggested that certain differentially expressed genes (DEGs)-such as MADS-box, homeobox family, and ethylene pathway genes-may be involved in the inflorescence meristem development. GO pathway enrichment analysis showed that the differentially transcribed genes enriched the representation of the carbohydrate metabolic pathway, which is critical for flower development. Overall, our findings revealed the conserved function of CmFL1 in controlling flowering time along with a novel function in regulating the number of florets.

Analyses of a chromosome-scale genome assembly reveal the origin and evolution of cultivated chrysanthemum.

Chrysanthemum (Chrysanthemum morifolium Ramat.) is a globally important ornamental plant with great economic, cultural, and symbolic value. However, research on chrysanthemum is challenging due to its complex genetic background. Here, we report a near-complete assembly and annotation for C. morifolium comprising 27 pseudochromosomes (8.15 Gb; scaffold N50 of 303.69 Mb). Comparative and evolutionary analyses reveal a whole-genome triplication (WGT) event shared by Chrysanthemum species approximately 6 million years ago (Mya) and the possible lineage-specific polyploidization of C. morifolium approximately 3 Mya. Multilevel evidence suggests that C. morifolium is likely a segmental allopolyploid. Furthermore, a combination of genomics and transcriptomics approaches demonstrate the C. morifolium genome can be used to identify genes underlying key ornamental traits. Phylogenetic analysis of CmCCD4a traces the flower colour breeding history of cultivated chrysanthemum. Genomic resources generated from this study could help to accelerate chrysanthemum genetic improvement.

The TIFY family protein CmJAZ1-like negatively regulates petal size via interaction with the bHLH transcription factor CmBPE2 in Chrysanthemum morifolium.

Petals are the second floral whorl of angiosperms, exhibiting astonishing diversity in their size between and within species. This variation is essential for protecting their inner reproductive organs and attracting pollinators for fertilization. However, currently, the genetic and developmental control of petal size remains unexplored. Chrysanthemum (Chrysanthemum morifolium) belongs to the Asteraceae family, the largest group of angiosperms, and the extraordinary diversity of petal size in chrysanthemums makes it an ideal model for exploring the regulation mechanism of petal size. Here, we reveal that overexpression of a JAZ repressor CmJAZ1-like exhibits decreased petal size compared to that of the wild-type as a result of repressed cell expansion. Through further in-depth exploration, we confirm an interaction pair between CmJAZ1-like and the bHLH transcription factor CmBPE2. The inhibition of CmBPE2 expression negatively regulates petal size by downregulating the expression of genes involved in cell expansion. Furthermore, CmJAZ1-like significantly reduced the activation ability of CmBPE2 on its target gene CmEXPA7 by directly interacting with it, thus participating in the regulation of petal size development in chrysanthemum. Our results will provide insights into the molecular mechanisms of petal size regulation in flowering plants.

DWARF AND ROBUST PLANT regulates plant height via modulating gibberellin biosynthesis in chrysanthemum.

YABBY (YAB) genes are specifically expressed in abaxial cells of lateral organs and determine abaxial cell fate. However, most studies have focused on few model plants, and the molecular mechanisms of YAB genes are not well understood. Here, we identified a YAB transcription factor in chrysanthemum (Chrysanthemum morifolium), Dwarf and Robust Plant (CmDRP), that belongs to a distinct FILAMENTOUS FLOWER (FlL)/YAB3 sub-clade lost in Brassicaceae. CmDRP was expressed in various tissues but did not show any polar distribution in chrysanthemum. Overexpression of CmDRP resulted in a semi-dwarf phenotype with a significantly decreased active GA3 content, while reduced expression generated the opposite phenotype. Furthermore, plant height of transgenic plants was partially rescued through the exogenous application of GA3 and Paclobutrazol, and expression of the GA biosynthesis gene CmGA3ox1 was significantly altered in transgenic plants. Yeast one-hybrid, luciferase, and chromatin immunoprecipitation-qPCR analyses showed that CmDRP could directly bind to the CmGA3ox1 promoter and suppress its expression. Our research reveals a nonpolar expression pattern of a YAB family gene in dicots and demonstrates it regulates plant height through the GA pathway, which will deepen the understanding of the genetic and molecular mechanisms of YAB genes.