Analyses of the bHLH gene family in Populus trichocarpa reveal roles of four PtbHLHs in regulating the drought stress response

As one of the largest families of transcription factors in plants, the basic helix-loop-helix (bHLH) transcription factor family regulates a wide range of functions in plants. However, little is known about the functions of bHLH family members in Populus trichocarpa during plant growth and in the response to drought stress. In our study, 190 PtbHLH genes were identified in the P. trichocarpa genome and classified into 21 groups. Analyses of microarray datasets showed that most PtbHLH members not only have multiple functions in poplar growth, but also respond rapidly to drought stress in the leaves or roots. We selected four genes, PtbHLH35, PtbHLH121, PtbHLH137, and PtbHLH152, which were highly expressed in leaves or roots under drought stress, for functional validation analyses. These genes encoded nucleus-localized bHLH transcription factors. Transient expression of PtbHLH35, PtbHLH121, and PtbHLH152 in P. trichocarpa improved drought tolerance by activating the antioxidant system to eliminate reactive oxygen species and reduce the degree of cell damage in the leaves under drought stress. Overexpression of PtbHLH137 improved drought tolerance by activating antioxidant enzymes in the roots to eliminate reactive oxygen species, and by increasing the abscisic acid content in the roots in response to drought stress. Together, our findings provide insights into the functions of PtbHLH family members in growth and in the response to drought.