Cucumber HD-ZIP III transcription factor CsPHB activates CsFAR3-like to regulate wax biosynthesis and cuticular water...

Transcriptional control of cuticular wax biosynthesis is important for plant adaptation to environmental stress. Although several transcription factors have been identified as key regulators of plant wax biosynthesis, the role of HD-ZIP III family members remains uncharacterized. Here, we demonstrate that the HD-ZIP III transcription factor PHABULOSA (CsPHB) positively regulates wax accumulation and cuticle barrier function in cucumber (Cucumis sativus L.). A gain-of-function mutation in CsPHB significantly increased the deposition of epicuticular wax crystals, resulting in greater cuticle thickness, lower cuticle permeability, and improved water retention capacity. Functional analyses of overexpression and CRISPR/Cas9-edited lines confirmed the role of CsPHB in regulating cuticular wax biosynthesis. Transcriptome profiling, promoter activation assays, and DNA-binding experiments further revealed that CsPHB directly activates fatty acyl-CoA reductase3-like (CsFAR3-like), which encodes a fatty acyl-CoA reductase participating in the primary alcohol biosynthesis pathway. Notably, CsFAR3-like was identified as a FAR family member that possesses primary alcohol-producing activity in cucurbits. Overexpression of CsFAR3-like led to greater wax accumulation and enhanced water retention, whereas knockout CsFAR3-like lines displayed lower wax accumulation and impaired cuticle function. Analysis of CsPHB gain-of-function × CsFAR3-like knockout double mutants revealed intermediate CsFAR3-like expression, wax accumulation, and water-deficit tolerance compared with the respective single mutants. Collectively, our findings uncover a previously uncharacterized CsPHB-CsFAR3-like transcriptional module that controls wax biosynthesis and cuticle function in cucumber, advancing our understanding of epidermal water retention mechanisms in this species.