SlGRF1 mediates gibberellin signaling to control cut-budding in tomato

Many dicotyledonous plants exhibit considerable developmental plasticity and are capable of regenerating new organs upon wounding. Leveraging this wound-induced cellular pluripotency, a simple, efficient, and genotype-independent "cut-dip-budding" transformation system has been developed in recent years. Although genetic transformation in dicotyledonous crops like tomato is still largely based on tissue culture methods, and research into their molecular regulatory mechanisms and regeneration factors is extensive, the mechanisms underlying cut-induced shoot regeneration (cut-budding) remain poorly understood. This study investigated the molecular basis of cut-budding in tomato, focusing on the role of GROWTH-REGULATING FACTOR1 (GRF1) and its association with gibberellin (GA) signaling. By combining single-cell RNA sequencing, time-course transcriptome analysis, and genetic validation, we elucidated the key stages of shoot regeneration and identified SlGRF1 as a critical regulator. SlGRF1 was revealed to be essential for shoot initiation, with its expression significantly upregulated during cut-budding. Functional characterization using CRISPR/Cas9 knockout mutants (grf1-cr) demonstrated that SlGRF1 is required for pluripotency acquisition and shoot formation. Additionally, GA signaling negatively regulated shoot initiation by repressing SlGRF1 expression. An exogenous GA treatment inhibited shoot regeneration, while a paclobutrazol (GA biosynthesis inhibitor) treatment had the opposite effect. Moreover, grf1-cr mutants were similar to GA1-treated samples in terms of transcriptional changes and phenotypes, further indicating that GA signaling represses SlGRF1 expression. A ChIP-seq analysis showed that SlGRF1 controls cut-budding by activating the expression of shoot apical meristem regulator-encoding genes, including NAM1, EPF4, and ER2. NAM1 overexpression rescued the defective regeneration of grf1-cr1 mutants, highlighting the role of NAM1 as a downstream effector of SlGRF1. The study findings further clarify the molecular mechanisms governing cut-budding in tomato.