The CLE9/CLE10 signaling peptides control de novo shoot regeneration in Arabidopsis

De novo shoot regeneration is widely exploited in plant biotechnology and genetic engineering. However, the deciding factors involved in this process remain largely elusive. Here, we elucidate the role of CLAVATA3/EMBRYO SURROUNDING REGION-RELATED 9/10 (CLE9/10) peptides during de novo shoot regeneration. Our data show that CLE9 and CLE10 are dynamically expressed in the callus. Exogenous application of the synthetic CLE9/10 peptide significantly represses de novo shoot regeneration. A similar inhibition of shoot organogenesis is also observed in CLE9 overexpression transgenic plants either driven by its native promoter or the inducible promoter. Conversely, cle9, cle10, and cle9 cle10 mutants display an increased number of adventitious shoots. These data jointly indicate that CLE9/10 peptides function redundantly as negative regulators in de novo shoot regeneration. Additionally, the significantly impaired shoot regeneration was found in transgenic plants expressing the CLE9G6T or CLE10G6T transgene, that might impair receptor binding, in which a glycine-to-threonine substitution was introduced in the conserved CLE motif. We further reveal that callus-expressed CLE9/CLE10 act through the BARELY ANY MERISTEM 1 (BAM1) receptor and restrict WUSCHEL (WUS) expression to control de novo shoot regeneration. Collectively, our work reveals the role and the molecular basis for the CLE9/CLE10 peptides in de novo shoot regeneration.