Chimeras of GROWTH-REGULATING FACTOR and GRF-INTERACTING FACTOR enhance leaf-based regeneration and transformation...

Efficient regeneration remains a major constraint for tomato (Solanum lycopersicum) transformation, particularly from leaf explants. Here, we evaluated chimeric GROWTH-REGULATING FACTOR (GRF)-GRF-INTERACTING FACTOR (GIF) proteins as regeneration enhancers and examined regulatory features underlying their activity. Fusion constructs based on Arabidopsis thaliana GRF5-GIF1 and their tomato homologs SlGRF4 and SlGIF1b markedly increased shoot regeneration from leaf explants, with similar enhancement observed in cotyledon-derived tissues, across three tomato cultivars. Regenerated shoots were able to form roots, indicating that GRF-GIF-mediated regeneration produced developmentally competent plantlets. Temporal expression profiling indicated that GRF-GIF transcript levels increased during regeneration and coincided with the induction of cytokinin- and auxin-associated regulators linked to meristem initiation. RNA-seq analyses revealed a shared early transcriptional program enriched for transcription factor activity and protein homeostasis, accompanied by broad repression of metabolism-related pathways. Despite strong phenotypic effects, GRF-GIF fusion proteins accumulated poorly in stable transformants. Transient expression assays suggested that their abundance is influenced by the ubiquitin-proteasome system, and mutational analyses identified lysine residues affecting protein stability, although stabilization did not further enhance regeneration. Together, these findings support the use of GRF-GIF chimeras as effective enhancers of tomato regeneration from leaf explants and highlight the contribution of coordinated regulation at the transcript and protein levels to their functional window during cellular reprogramming.