Abstract: Somatic embryogenesis (SE) enables somatic cells to develop directly into embryos. SE is a major approach of regeneration, but recalcitrance to SE has become one of the main obstacles to biotechnology-aided breeding, especially for perennial woody plants. Citrus is one of the most important fruit crops in the world, and glycerol has long been used to induce SE from the embryogenic callus (EC) of citrus. Recently, we reported that CsIAA4-mediated repression of auxin signaling plays a critical role in glycerol-induced citrus SE, but the downstream signaling cascade remains to be elucidated. In this study, the HD-Zip transcription factor CsHAT14 was identified as a key downstream regulator of auxin signaling in citrus SE. CsARF5 directly promoted CsHAT14 expression, which repressed SE through suppression of critical regeneration-related genes (CsDOF3.4 and CsWOX13) and the auxin efflux gene CsPILS5. CsIAA4 interacted with CsARF5, and this interaction attenuated CsARF5-mediated transcriptional activation of CsHAT14, thereby de-repressed CsHAT14- directly suppressed genes including CsDOF3.4, and thus promoted SE. Knockdown of CsDOF3.4 resulted in downregulation of cell cycle-related genes and impaired SE. Our findings established the CsIAA4–CsARF5 and CsHAT14–CsDOF3.4 modules-mediated auxin signaling cascade that coordinates citrus SE, which advanced our understanding of the mechanisms underlying SE and supported improvement of regeneration efficiency in citrus biotechnology applications.

Key words: auxin signaling, citrus, CsDOF3.4, CsHAT14, somatic embryogenesis