Plant-specific TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) proteins play critical roles in plant development and stress responses; however, their functions in chrysanthemum (Chrysanthemum morifolium) have not been well-studied. In this study, we isolated and characterized the chrysanthemum TCP transcription factor family gene CmTCP13, a homolog of AtTCP13. This gene encoded a protein harboring a conserved basic helix–loop–helix motif, and its expression was induced by salinity stress in chrysanthemum plants. Subcellular localization experiments showed that CmTCP13 localized in the nucleus. Sequence analysis revealed the presence of multiple stress- and hormone-responsive cis-elements in the promoter region of CmTCP13. The heterologous expression of CmTCP13 in Arabidopsis plants enhanced their tolerance to salinity stress. Under salinity stress, CmTCP13 transgenic plants exhibited enhanced germination, root length, seedling growth, and chlorophyll content and reduced relative electrical conductivity compared with those exhibited by wild-type (WT) plants. Moreover, the expression levels of stress-related genes, including AtSOS3, AtP5CS2, AtRD22, AtRD29A, and AtDREB2A, were upregulated in CmTCP13 transgenic plants than in WT plants under salt stress. Taken together, our results demonstrate that CmTCP13 is a critical regulator of salt stress tolerance in plants.

The sucrose nonfermenting 1-related protein kinase (SnRK) plays an important role in responding to abiotic stresses by phosphorylating the target protein to regulate various signaling pathways. However, little is known about the characteristics, evolutionary history, and expression patterns of the SnRK family in black raspberry (Rubus occidentalis L.) or other Rosaceae family species. In this study, a total of 209 SnRK genes were identified in 7 Rosaceae species and divided into 3 subfamilies (SnRK1, SnRK2, and SnRK3) based on phylogenetic analysis and specific motifs. Whole-genome duplication (WGD) and dispersed duplication (DSD) were considered to be major contributions to the SnRK family expansion. Purifying selection was the primary driving force in the SnRK family evolution. The spatial expression indicated that the RoSnRK genes may play important roles in different tissues. In addition, the expression models of 5 RoSnRK2 genes in response to abiotic stresses were detected by qRT-PCR. The proteins encoded by RoSnRK2 genes localize to the cytoplasm and nucleus in order to perform their respective functions. Taken together, this study provided an analysis of the SnRK gene family expansion and evolution, and contributed to the current knowledge of the function of 5 RoSnRK2 genes, which in turn expanded understanding of the molecular mechanisms of black raspberry responses to abiotic stress.

The holly Ilex dabieshanensis K. Yao & M. B. Deng, a tree endemic to the Dabieshan Mountains region in China, is a commonly used landscaping plant. Like other crops, its growth is affected by salt stress. The molecular mechanism underlying salt tolerance in holly is still unclear. In this study, we used NaCl treatment and RNA sequencing (RNA-seq) at different times to identify the salt stress response genes of holly. A total of 4775 differentially expressed genes (DEGs) were identified. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the DEGs obtained at different salt treatment times (3, 6, 9, 12, and 24 h), as compared to control (ck, 0 h), showed that plant hormone signal transduction and carotenoid biosynthesis were highly enriched. The mechanism by which holly responds to salt stress involves many plant hormones, among which the accumulation of abscisic acid (ABA) and its signal transduction may play an important role. In addition, ion homeostasis, osmotic metabolism, accumulation of antioxidant enzymes and nonenzymatic antioxidant compounds, and transcription factors jointly regulate the physiological balance in holly, providing important guarantees for its growth and development under conditions of salt stress. These results lay the foundation for studying the molecular mechanisms of salt tolerance in holly and for the selection of salt-tolerant varieties.

Chrysanthemum morifolium Ramat. is an important ornamental plant, holding dual economic value as a medicinal and edible plant. Jinsi Huangju is a popular healthy tea drink prepared from the large and elegant shaped flowers of C. morifolium. However, the suboptimal accumulation of bioactive flavonoids during conventional harvest (full bloom stage) limits its commercial potential. To elucidate the molecular mechanisms governing flavonoid biosynthesis in Jinsi Huangju flowers and identify key genetic regulators for metabolic engineering, we performed integrated metabolomic and transcriptomic analyses of flowers at distinct developmental stages using ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) and RNA-seq. Differential metabolites were screened, and candidate genes were validated via transient transformation assays. Among 2146 identified metabolites, flavonoids were the predominant differential compounds, with accumulation patterns being strongly stage dependent. Thirty-eight flavonoid biosynthetic genes and key transcription factors from the MYB, bHLH, and WD40 families exhibited dynamic expression. The CmMYB8a was confirmed as a positive regulator of flavonoid biosynthesis through transient overexpression. This study deciphers the stage-specific flavonoid accumulation in Jinsi Huangju and identifies CmMYB8a as a pivotal regulatory target. Our findings provide genetic resources for breeding high-flavonoid cultivars via molecular design.

Chrysanthemum (Chrysanthemum morifolium) is a globally significant ornamental plant, whose growth, development, and ornamental quality are frequently impaired by salt stress. DOF (DNA-binding with one finger) family transcription factors extensively act as crucial regulators in medicating reactions to environmental pressures on plants. But their specific functions in regulating salt stress tolerance in chrysanthemum still remain largely elusive and require further investigation. Here, we isolated CmDOF2, a DOF family transcription factor from chrysanthemum, whose expression was up-regulated in chrysanthemums under salt stress. Functional analysis demonstrated that CmDOF2 functions as a nuclear-localized transcriptional activator. Comprehensive phenotypic and physiological characterization showed that heterologous expression of CmDOF2 in Arabidopsis thaliana conferred markedly increased salt stress tolerance, as reflected by higher chlorophyll, leaf relative water, and proline content; lower leaf relative electric conductivity and malondialdehyde content; and increased activities of superoxide dismutase, peroxidase, and catalase. Furthermore, qRT-PCR analyses confirmed that stable expression of CmDOF2 in Arabidopsis led to increased transcript levels of key salt-responsive genes, including stress marker genes (AtRD29A, AtRD29B), components of the SOS signaling pathway (AtSOS1, AtSOS2, AtSOS3), genes involved in osmotic adjustment (AtP5CS1, AtP5CS2), and genes encoding antioxidant enzymes (AtSOD1, AtPOD34, AtCAT3). Collectively, our data demonstrate that CmDOF2 serves as a nuclear-localized transcriptional regulator with activation activity and positively regulates salt stress tolerance by mediating the transcript levels of stress-related genes in multiple signaling pathways.