The bHLH transcription factor CsPIF4 positively regulates high temperature-induced hypocotyl elongation in cucumber

High temperature-induced hypocotyl elongation is a typical thermomorphogenesis trait that may significantly affect early seedling growth and subsequent crop yield. The ambient temperature and endogenous auxin are two critical factors that regulate hypocotyl growth. However, the mechanism of temperature and auxin integration in horticultural plants remains poorly understood. In this study, the roles of the basic helix-loop-helix transcription factor CsPIF4 in regulating auxin biosynthesis genes and the auxin content in the hypocotyl of cucumber (Cucumis sativus L.) seedlings under high temperature were investigated. qRT-PCR and in situ hybridization analysis revealed that expression of CsPIF4 was enhanced in the epidermis and vascular bundles in the hypocotyl of cucumber seedlings in response to high temperature. qRT-PCR and HPLC analysis showed that CsPIF4 positively regulated transcription of the auxin biosynthesis gene CsYUC8 and the auxin content in the hypocotyl under high temperature (35 °C). The CRISPR/Cas9-mediated knockout of CsPIF4 resulted in a shorter hypocotyl compared with that of the wild type, together with decreased expression of CsYUC8 and lower auxin content in response to high temperature. Furthermore, biochemical assays showed that CsPIF4 could bind directly to the G-box motif of the CsYUC8 promoter and thereby activate CsYUC8 expression. These findings provide insight into the molecular mechanism of high temperature-mediated hypocotyl elongation in cucumber.