Genome-wide identification of the bHLH transcription factor family and the regulatory roles of PbbHLH74 in response to drought stress in Phoebe bournei.

Phoebe species constitute a large portion of subtropical forestry, which are key players in biomass resources. However, abiotic stresses such as drought stress severely limit the growth and development of P. bournei, and even lead to its death. It has been shown that basic helix-loop-helix (bHLH) as the second largest transcription factor family plays essential roles in response to multiple stresses in plants. However, little information of bHLH family is available in P. bournei. In this study, 130 PbbHLHs were identified and classified into 24 subfamilies. Then, the bHLH domain, conserved motifs and gene structures, evolutionary patterns and protein structural features were probed. The expression levels of 17 PbbHLHs were differentially induced by PEG and ABA by RT-qPCR analysis, indicating that they may be involved in drought stress response. Characterization of the drought candidate gene PbbHLH74 showed that it was transcriptionally active and localized in the nucleus. Heterologous transformation of PbbHLH74 into yeast improved cellular tolerance to drought stress. Meanwhile, overexpression of PbbHLH74 in Arabidopsis showed higher seed germination, plant biomass and expression levels of stress-related genes under drought conditions. Through the hairy root technique, overexpression of PbbHLH74 in P. bournei improved drought tolerance by enhancing root development and expression levels of genes involved in ABA-dependent and ROS scavenging pathways. Moreover, PbbHLH74 might positively regulate the expression of PbPOD by Y1H and dual-luciferase reporter assays. Overall, these results elucidated the structure and evolution of the PbbHLH family, in which PbbHLH74 could be applied to molecular assisted breeding for drought tolerance in P. bournei.