Unraveling the molecular framework of de novo shoot organogenesis in Arabidopsis thaliana

Plant regeneration can be defined as the ability to repair tissues and reconstruct organs upon wounding or in vitro explant cultivation. This phenomenon is exploited in various biotechnological applications, but many species are recalcitrant to existing tissue culture methods. Hence, this work investigates shoot regeneration from root explants in the model plant Arabidopsis thaliana, which can be achieved by a two-step protocol involving pre-incubation on auxin-rich callus-inducing medium, followed by transfer to cytokinin- rich shoot induction medium. By means of a genome-wide association study, multi-omics data integration via biological networks, a phosphoproteome analysis, mutational and chemical genetics, molecular factors were identified that control de novo organogenesis. Among these are established master regulators such as AHK4, WUS, and AGO1, but also novel candidates, including POE1;19, EDA40, DOF4.4, DREB2A, MYB118, RD2, PVA11, and AVT1C. Enriched ontologies relate to stress responses, hormone signaling, meristem maintenance, shoot and root morphogenesis, epigenetics, endomembrane dynamics, etc. Intriguingly, the proposed factors are regulated at various omic levels (e.g., DNA sequence variation, differential transcription, and post-translational modifications), depending on their position in the molecular hierarchy. The results further reveal that regeneration is highly variable and many individual gene contributions are specific for a particular experimental system, urging future research in the field to consider multiple incubation conditions and genetic backgrounds. Finally, our data provide prime targets for molecular marker development and support rationalized protocol design.