The apetala/ethylene responsive factor (AP2/ERF) family is one of the largest plant-specific transcription factors and plays a vital role in plant development and response to stress. The apetala 2.4 (RAP2.4) gene is a member of the AP2/ERF family. In this study, ClRAP2.4 cDNA fragment with 768 bp open reading frame was cloned and the resistance of ClRAP2.4 overexpression to low temperature was investigated to understand whether RAP2.4 is involved in low-temperature stress in chrysanthemum (Chrysamthemum lavandulifolium). Phylogenetic analysis showed that ClRAP2.4 belonged to the DREB subfamily and was most closely related to AT1G22190. ClRAP2.4 was localised in cell nucleus and promotes transcriptional activation in yeast. In addition, ClRAP2.4 was transformed by using the Agrobacterium-mediated leaf disc method, and four overexpression lines (OX-1, OX-2, OX-7, and OX-8) were obtained. The activities of superoxide dismutase and peroxidase, and proline content in leaves in the four overexpression line were higher than those in the wild type (WT), whereas the electrical conductivity and malondialdehyde content were decreased, indicating that the tolerance of plants with ClRAP2.4 overexpression to cold stress was increased. RNA-Seq showed 390 differentially expressed genes (DEGs) between the transgenic and WT plants(229 upregulated, 161 downregulated). The number of ABRE, LTR, and DRE cis-elements in the promoters of DEGs were 175, 106, and 46, respectively. The relative expression levels of ClCOR, ClFe/MnSOD, ClPOD, ClNCL, ClPLK, ClFAD, and ClPRP in the transgenic plants were higher than those in WT plants at low temperatures. These data suggest that ClRAP2.4 may increase chrysanthemum tolerance to cold stress.

Both the presence of, and the important contribution to growth and development made by TCP transcription factors, have been established in various plant species. Here, a TCP4 homolog isolated from Chrysanthemum nankingense was shown to be more strongly transcribed in the diploid than in the autotetraploid form of the species. CnTCP4 was shown to encode a member of the class II TCP family and to be transcribed most strongly in the leaf and ligulate flowers. Its transcription was found to be substantially inhibited by spraying the plant with the synthetic cytokinin 6-benzylaminopurine. The transient expression of CnTCP4 in onion epidermal cells showed that its product localized to the nucleus, and a yeast one hybrid assay suggested that its product had transcriptional activation ability. The constitutive expression of CnTCP4 in fission yeast suppressed cell proliferation, inducing the formation of longer and a higher frequency of multinuclated cells. Its constitutive expression in Arabidopsis thaliana reduced the size of the leaves. The presence of the transgene altered the transcription of a number of cell division-related genes. A yeast one hybrid assay identified a second TCP gene (CnTCP2) able to interact with the CnTCP4 promoter. A transient expression experiment in Nicotiana benthamiana leaves showed that CnTCP2 was able to activate the CnTCP4 promoter. Like CnTCP4, CnTCP2 was shown to encode a member of the class II TCP family, to be transcribed most strongly in the leaf and ligulate flowers, and to be suppressed by exogenous 6-benzylaminopurine treatment. The CnTCP2 protein also localized to the nucleus, but had no transcriptional activation ability. Its constitutive expression in A. thaliana had similar phenotypic consequences to those induced by CnTCP4.