A CLE–RLK–LBD signaling module promotes de novo shoot regeneration in plants

Tissue culture recalcitrance limits plant transformation and genome editing for crop improvement. The development of new, generic factors that enhance plant regeneration efficiency could alleviate this bottleneck. Through precursor gene overexpression, exogenous peptide application, and loss-of-function studies, we show here that CLAVATA3/ESR-RELATED 41 (CLE41), CLE42, CLE44, and CLE46, collectively referred to as TDIF-related genes, function redundantly to promote de novo shoot regeneration. CLE41, CLE42, and CLE44 are abundantly and dynamically expressed in pluripotent callus, whereas CLE46 is shoot-induction medium-activated and specifically expressed at explant wound sites. TDIF-related peptides act through PHLOEM INTERCALATED WITH XYLEM/TDIF RECEPTOR (PXY/TDR)-subfamily members to activate WUSCHEL (WUS)-RELATED HOMEOBOX 14 (WOX14) and enhance shooting capacity. In addition, we find that LATERAL ORGAN BOUNDARIES DOMAIN4 (LBD4) is highly expressed throughout the callus and its expression increases upon TDIF upregulation in a PXY/PXL-dependent manner. Consistently, LBD4 overexpression notably improves regeneration efficiency, whereas its loss decreases regeneration. TDIF-enhanced shoot regeneration is significantly suppressed in lbd4 mutants, indicating that LBD4 is required for TDIF signaling in shoot regeneration enhancement. Furthermore, WOX14 directly activates LBD4 to promote de novo shoot regeneration. Exogenous application of synthetic TDIF-related peptides substantially increases shooting capacity in the recalcitrant crop species Sorghum bicolor L., offering a convenient new avenue to enhance de novo shoot regeneration in crops. In conclusion, we demonstrate that Arabidopsis TDIF-PXY/PXL ligand-receptor pairs markedly boost de novo shoot regeneration through WOX14-potentiated LBD4 induction. Our findings elucidate the molecular basis for a novel function of TDIF-related peptides and their receptors in hormone-driven shoot regeneration and demonstrate their practical application in improving adventitious shoot regeneration in a recalcitrant plant species.