A major constraint affecting the quality and productivity of chrysanthemum is the unusual period of low temperature occurring during early spring, late autumn, and winter. Yet, there has been no systematic investigation on the genes underlying the response to low temperature in chrysanthemum. Herein, we used RNA-Seq platform to characterize the transcriptomic response to low temperature by comparing different transcriptome of Chrysanthemum nankingense plants and subjecting them to a period of sub-zero temperature, with or without a prior low temperature acclimation. Six separate RNA-Seq libraries were generated from the RNA samples of leaves and stems from six different temperature treatments, including one cold acclimation (CA), two freezing treatments without prior CA, two freezing treatments with prior CA and the control. At least seven million clean reads were obtained from each library. Over 77% of the reads could be mapped to sets of C. nankingense unigenes established previously. The differentially transcribed genes (DTGs) were identified as low temperature sensing and signalling genes, transcription factors, functional proteins associated with the abiotic response, and low temperature-responsive genes involved in post-transcriptional regulation. The differential transcription of 15 DTGs was validated using quantitative RT-PCR. The large number of DTGs identified in this study, confirmed the complexity of the regulatory machinery involved in the processes of low temperature acclimation and low temperature/freezing tolerance.

Chrysanthemum is among the top ten traditional flowers in China, and one of the four major cut flowers in the world, but the growth of chrysanthemum is severely restricted by high temperatures which retard growth and cause defects in flowers. DREB (dehydration-responsive element-binding) transcription factors play important roles in the response to abiotic and biotic stresses. However, whether the DREB A-6 subgroup is involved in heat tolerance has not been reported conclusively. In the present study, CmDREB6 was cloned from chrysanthemum (Chrysanthemum morifolium) ‘Jinba’. CmDREB6, containing a typical AP2/ERF domain, was classed into the DREB A-6 subgroup and shared highest homology with Cichorium intybus L. CiDREB6 (73%). CmDREB6 was expressed at its highest levels in the leaf. The CmDREB6 protein localized to the nucleus. Based on the yeast one hybrid assay, CmDREB6 showed transcription activation activity in yeast, and the transcriptional activation domain was located in the 3 ‘end ranging from 230 to 289 amino acids residues. CmDREB6 overexpression enhanced the tolerance of chrysanthemum to heat. The survival rate of two transgenic lines was as high as 85%, 50%, respectively, in contrast to 3.8% of wild-type (WT). Over-expression of CmDREB6 promoted the expression of CmHsfA4, CmHSP90, and the active oxygen scavenging genes CmSOD and CmCAT. In this study, DREB A-6 subgroup gene CmDREB6 was cloned from chrysanthemum ‘Jinba’. Overexpression of CmDREB6 enhanced heat tolerance of chrysanthemum by regulating genes involved in the heat shock response and ROS homeogenesis.

Trihelix transcription factors are thought to feature a typical DNA-binding trihelix (helix-loop-helix-loop-helix) domain that binds specifically to the GT motif, a light-responsive DNA element. Members of the trihelix family are known to function in a number of processes in plants. Here, we characterize 20 trihelix family genes in the important ornamental plant chrysanthemum (Chrysanthemum morifolium). Based on transcriptomic data, 20 distinct sequences distributed across four of five groups revealed by a phylogenetic tree were isolated and amplified. The phylogenetic analysis also identified four pairs of orthologous proteins shared by Arabidopsis and chrysanthemum and five pairs of paralogous proteins in chrysanthemum. Conserved motifs in the trihelix proteins shared by Arabidopsis and chrysanthemum were analyzed using MEME, and further bioinformatic analysis revealed that 16 CmTHs can be targeted by 20 miRNA families and that miR414 can target 9 CmTHs. qPCR results displayed that most chrysanthemum trihelix genes were highly expressed in inflorescences, while 20 CmTH genes were in response to phytohormone treatments and abiotic stresses. This work improves our understanding of the various functions of trihelix gene family members in response to hormonal stimuli and stress.

Chrysanthemum, one of the most important commercial ornamental crops, is susceptible to salinity, which limits its cultivation and application in coastal and inland saline areas. Grafting is widely used to improve the salt tolerance of horticultural crops, but the mechanisms of grafted chrysanthemum responses to salt stress remain unclear. In this study, we showed that heterografted chrysanthemums with Artemisia annua as rootstock exhibited increased salt tolerance compared with self-grafted and self-rooted chrysanthemums. Under high salt stress, the roots of heterografted chrysanthemums enrich Na⁺, resulting in a reduction of Na⁺ toxicity in the scion, with only a small amount of Na⁺ being transported to the leaves. On the other hand, the roots of heterografted chrysanthemums alleviated high Na⁺ stress via enhanced catalase enzyme activity, downregulation of the expression of reactive oxygen species (ROS) accumulation-related genes, massive accumulation of soluble sugars and proline, and upregulation of the expression of heat shock protein-related genes to enhance salt tolerance. In addition, the leaves of heterografted chrysanthemums respond to low Na⁺ stress by increasing peroxidase enzyme activity and soluble sugar and proline contents, to maintain a healthy state. However, self-grafted and self-rooted plants could not integrate ROS, soluble sugars, and proline in response to salt stress, and thus exhibited a salt-sensitive phenotype. Our research reveals the mechanisms underlying the increased salt tolerance of heterografted chrysanthemums and makes it possible to have large-scale cultivation of chrysanthemums in saline areas.

The green organs, especially the leaves, of many Compositae plants possess characteristic aromas. To exploit the utility value of these germplasm resources, the constituents, mainly volatile compounds, in the leaves of 14 scented plant materials were qualitatively and quantitatively compared via gas chromatography-mass spectrometry (GC-MS). A total of 213 constituents were detected and tentatively identified in the leaf extracts, and terpenoids (especially monoterpene and sesquiterpene derivatives), accounting for 40.45–90.38% of the total compounds, were the main components. The quantitative results revealed diverse concentrations and compositions of the chemical constituents between species. Principal component analysis (PCA) showed that different groups of these Compositae plants were characterized by main components of α-thujone, germacrene D, eucalyptol, β-caryophyllene, and camphor, for example. On the other hand, cluster memberships corresponding to the molecular phylogenetic framework, were found by hierarchical cluster analysis (HCA) based on the terpenoid composition of the tested species. These results provide a phytochemical foundation for the use of these scented Compositae plants, and for the further study of the chemotaxonomy and differential metabolism of Compositae species.