Identification and characterization of AnICE1 and AnCBFs involved in cold tolerance from Ammopiptanthus nanus

The ICE-CBF-COR pathway plays a vital role in improving the cold tolerance of plants. As an evergreen small shrub, Ammopiptanthus nanus has a high tolerance to cold stress because of its special growth conditions. Regrettably, no cold-responsive genes in the ICE-CBF-COR pathway have been reported in A. nanus. In the current study, we isolated AnICE1, AnCBF1, and AnCBF2 in A. nanus and analyzed their sequence structure. Evolutionary analysis indicated that these genes are most closely related to those from Ammopiptanthus mongolicus, Glycine max, Spatholobus suberectus, and Cajanus cajan, all belonging to the Fabaceae. Expression analysis showed that the expression levels of these genes were induced under cold stress and treatment with several plant hormones. As a critical upstream regulator in the ICE-CBF-COR pathway, the function of AnICE1 was further identified. The subcellular localization indicated that AnICE1 is predominantly localized in the plasma membrane and less in the nucleus. Overexpression of AnICE1 in Arabidopsis thaliana improved seed germination and growth of transgenic seedlings during cold stress. Moreover, some physiological indices such as relative electrical conductivity, contents of proline and malondialdehyde, catalase activity, and Nitro Blue tetrazolium and 3.3'-diaminobenzidine staining were investigated by transient expression in A. nanus seedlings and stable overexpression in A. thaliana. These results indicated that AnICE1 enhanced cold tolerance in A. nanus and transgenic A. thaliana. This study is significant for understanding the cold-resistant mechanism of ICE and CBF genes in A. nanus.