Genome-wide identification of Osmanthus fragrans bHLH transcription factors and their expression analysis in response to abiotic stress

Sweet osmanthus (Osmanthus fragrans) is a widely used evergreen aromatic ornamental tree with poor tolerance to low temperatures. The basic helix-loop-helix (bHLH) transcription factor protein family, which is the second largest protein family in plants, has been found to play essential roles in the response to multiple abiotic and biotic stresses, especially cold stress. However, the bHLH gene family in sweet osmanthus has not yet been analyzed. In this study, 206 OfbHLH genes, which were classified into 25 subfamilies, were successfully identified from recently released whole-genome sequence data. Segmental duplication events had contributed mainly to the expansion of the OPHLH gene family and all these duplicated gene pairs had experienced purifying selection. The transcriptome data exhibited different expression levels of OfbHLH genes in various tissues. Quantitative real-time PCR results indicated that the expression patterns of most Mb, III(d + e), Nc, DC, and XII subfamilies could be up- or down-regulated under cold stress, and, among them, Mb, M(d + e), and part of the XII subfamily participated in the response to multiple abiotic stresses. Six candidate OfbHLH genes (OfbHLH120/128/133/163/164/191), which were significantly up- or down-regulated during the cold stress process, were identified. Further studies indicated that OfbHLH120/128/163 had transcriptional activation activity and were located in the nucleus, implying that these genes are involved in the regulation of expression of cold tolerance genes. This study provides a solid foundation for the functional analysis of the OfbHLH gene family members involved in cold stress response and could be useful in the breeding of sweet osmanthus with improved cold tolerance.