Successive grafting reprograms vegetative phase transition in black locust (Robinia pseudoacacia L.), a woody crop species

Rejuvenation of mature woody plants is critical for restoring regenerative capacity and improving clonal propagation efficiency, yet effective induction strategies and their regulatory mechanisms remain poorly understood. This study aimed to determine whether successive grafting onto juvenile root-cutting rootstocks can induce systemic rejuvenation in mature black locust and to elucidate the underlying physiological and molecular mechanisms. Apical scions collected from mature trees were subjected to two successive grafting cycles onto juvenile root-cutting rootstocks, generating first- and second-generation grafted seedlings, which were compared with mature trees and juvenile root-cutting plantlets. Morphological, anatomical, physiological, transcriptomic, small RNA, and global DNA methylation analyses were conducted to evaluate rejuvenation-related changes. Successive grafting progressively restored juvenile traits, including increased leaf area, enhanced trichome density, reduced leaf thickness, and elongation of spines. Physiological analyses revealed decreased protein, phenolic, and malondialdehyde contents, together with a marked increase in net photosynthetic rate. Molecular analyses showed significant upregulation of microRNA156, repression of microRNA172, and a pronounced reduction in global DNA methylation levels during rejuvenation. Weighted gene co-expression network analysis identified RpMYC2 as a central transcriptional hub whose expression increased across grafting generations and was accompanied by elevated endogenous jasmonic acid accumulation and coordinated induction of jasmonate-related genes. Heterologous overexpression of RpMYC2 in Arabidopsis thaliana delayed flowering and suppressed AtFT expression, indicating a conserved role in age-related developmental regulation. These results demonstrate that successive grafting onto juvenile root-cutting rootstocks is an effective strategy for inducing rejuvenation in mature black locust and identify RpMYC2 as a key regulator linking jasmonate signaling to developmental phase reversal. • Successive grafting onto juvenile root-cutting rootstocks effectively induces rejuvenation in mature black locust. • Rejuvenated scions exhibit juvenile traits: altered leaf morphology, increased trichomes, and enhanced photosynthesis. • Rejuvenation involves upregulated microRNA156, downregulated microRNA172, and reduced global DNA methylation. • RpMYC2 is identified as a key regulator; its heterologous overexpression delays flowering in Arabidopsis .