Overexpression of ClRAP2.4 in Chrysanthemum enhances tolerance to cold stress

Abstract

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.