DgHDA6 enhances the cold tolerance in chrysanthemum by improving ROS scavenging capacity.

Histone deacetylases have been demonstrated to play an important role in responding to low-temperature stress, but the related response mechanism in chrysanthemum remains unclear. In this study, we isolated a cold-induced gene, DgHDA6, from chrysanthemum (Chrysanthemum morifolium Ramat). DgHDA6 contains 474 amino acids and shares a typical deacetylation domain with RPD3/HDA1 family members. The overexpression of DgHDA6 enhanced cold resistance in chrysanthemums. After low-temperature stress, the overexpression lines showed a higher survival rate. The contents of proline, soluble proteins and sugars, and the activities of antioxidant enzymes were significantly increased while the contents of H2O2, O2- and MDA were lower. Moreover, cold-stress-responding genes such as DgCuZnSOD, DgCAT, DgP5CS, and DgFAD were upregulated after cold stress. These results suggest that the overexpression of DgHDA6 can improve cold tolerance in chrysanthemum by enhancing ROS scavenging capacity.

Lysine malonylation of DgnsLIPID TRANSFER PROTEIN1 at the K81 site improves cold resistance in chrysanthemum.

Lysine malonylation (Kmal) is a recently discovered posttranslational modification, and its role in the response to abiotic stress has not been reported in plants. In this study, we isolated a nonspecific lipid transfer protein, DgnsLTP1, from chrysanthemum (Dendranthema grandiflorum var. Jinba). Overexpression and CRISPR-Cas9-mediated gene editing of DgnsLTP1 demonstrated that the protein endows chrysanthemum with cold tolerance. Yeast 2-hybrid, bimolecular fluorescence complementation, luciferase complementation imaging, and coimmunoprecipitation experimental results showed that DgnsLTP1 interacts with a plasma membrane intrinsic protein (PIP) DgPIP. Overexpressing DgPIP boosted the expression of DgGPX (glutathione peroxidase), increased the activity of GPX, and decreased the accumulation of reactive oxygen species (ROS), thereby enhancing the low-temperature stress tolerance of chrysanthemum, while the CRISPR-Cas9-mediated mutant dgpip inhibited this process. Transgenic analyses in chrysanthemum showed that DgnsLTP1 improves the cold resistance of chrysanthemum in a DgPIP-dependent manner. Moreover, Kmal of DgnsLTP1 at the K81 site prevented the degradation of DgPIP in Nicotiana benthamiana and chrysanthemum, further promoted DgGPX expression, enhanced GPX activity, and scavenged excess ROS produced by cold stress, thereby further enhancing the cold resistance of chrysanthemum.