2,4-Dichlorophenoxyacetic acid (2,4-D) is widely used to induce somatic embryogenesis (SE), yet the concentration-dependent molecular mechanisms governing this process remain poorly resolved. Here, we investigated the morphological responses and proteomic changes in Carica papaya zygotic embryos exposed to a range of 2,4-D concentrations (0-200 µM) to identify regulatory pathways underlying embryogenic competence. Morphological assays showed that 20 µM 2,4-D was the only treatment capable of inducing embryogenic callus by day 14, whereas supra-optimal doses delayed or impaired SE. A label-free quantitative proteomic analysis performed at seven days after induction identified 1168 proteins, of which 726 were differentially accumulated at the onset of callus induction. Clustering revealed six dose-dependent accumulation patterns, with high 2,4-D concentrations broadly repressing proteins associated with auxin signaling, proteasome-mediated turnover, methylation-related metabolism, proton transport and chromatin remodeling. In contrast, moderate 2,4-D levels preserved proton pump activity, maintained SCF-AUX/IAA signaling, supported GH3-mediated auxin conjugation, sustained proteasomal components and promoted SAM/SAHH-dependent methylation, collectively creating a molecular environment permissive to cellular reprogramming. At 200 µM 2,4-D, the proteomic profile showed a coordinated repression of biosynthetic, redox and developmental pathways, as demonstrated by the under-enrichment of metabolic and energy-associated GO terms. Functional enrichment also revealed only limited activation of stress-associated categories, with hypoxia emerging as the sole enriched BP term. Together, GO and KEGG analyses indicate that supra-optimal auxin simultaneously suppresses core metabolic pathways and fails to activate broader adaptive stress programs, disrupting the interconnected functional modules required for embryogenic reprogramming. These findings demonstrate that SE in C. papaya is governed by a narrow auxin window that balances hormonal signaling, proteostasis, metabolic activity and chromatin regulation, while excessive auxin shifts the proteome toward a metabolically repressed state. This work establishes a mechanistic framework for optimizing auxin-based SE protocols in C. papaya. Optimal 2,4-D concentration promotes embryogenic callus formation by coordinating metabolic, proteomic, and epigenetic networks, while higher doses trigger stress and inhibit somatic embryogenesis.
Pacheco et al. (Wed,) studied this question.