Genome-wide identification of cold-tolerance genes and functional analysis of IbbHLH116 gene in sweet potato

Sweet potato (Ipomoea batatas L.) originated from South America; therefore, it is vulnerable to low temperature. Here, the evolutionary analysis of 22 cold-responsive genes in 35 plant species revealed that the identified MYCtype basic helix-loop-helix (bHLH) transcription factors exhibit diverse structures. We found that the number of bHLH gene family members was significantly lower than that of cold-tolerant species. We further systematically evaluated the gene structure, promoter analysis, synteny analysis, and expression pattern of 28 bHLH gene family members in sweet potato. The basic helix-loop-helix protein 116 (IbbHLH116) has the closest phylogeny to the AtICE1 protein of A. thaliana. However, the IbbHLH116 protein from cold-tolerant variety FS18 showed a 37.90% of sequence homology with AtICE1 protein. Subcellular localization analysis showed that IbbHLH116 is localized in the nucleus. The transcripts of IbbHLH116 were highly accumulated in cold-tolerant genotype FS18, particularly in new leaves and stems, compared to the cold-sensitive genotype NC1 under cold stress. Overexpression of IbbHLH116 in the wild type (Col-0) A. thaliana significantly enhanced cold tolerance in transgenic plants by regulating activities of oxidative protective enzymes, such as peroxidase (POD), superoxide dismutase (SOD), and the contents of malondialdehyde (MDA), proline and soluble proteins. Moreover, overexpression of IbbHLH116 in ice1 mutant A. thaliana fully rescued the cold-sensitive phenotype by promoting the expression of C-repeat binding factors 3 (CBF3). Overexpression of IbbHLH116 in the sweet potato callus also induced the expression of CBF3 under low temperature. These results imply that IbbHLH116 can perform the function of the ICE1 gene in conferring cold tolerance in sweet potato.