Integrative Omics and Genetic Engineering Approaches to Decipher the Molecular Basis of Salt and Osmotic Stress in Plant Tolerance Models

Climate change is shrinking arable land and tightening water and soil quality constraints, making osmotic and saline stress primary threats to crop productivity. This thesis integrates physiology, multi-omics, and targeted genome engineering to elucidate mechanisms of stress tolerance and identify deployable gene targets. Two complementary models were used: (i) a diploid–tetraploid comparison in Medicago sativa and Solanum commersonii to test how whole-genome duplication (WGD) reshapes stress responses; and (ii) an in-vitro Solanum tuberosum (cv. Désirée) cell system designed to highlight the differences in molecular response between abrupt versus gradual adaptation to PEG-imposed water deficit. These activities were carried out at PlantaLab, in the Department of Pharmacy of the University of Salerno. The Alfalfa diploid versus tetraploid model proved to be more suitable for further omics investigations, with a more divergent response in phenotypic and physiological to salt stress. Comparative RNA-seq revealed that ploidy explained more variance than treatment: thousands of DEGs distinguished 4x from 2x under both control and salt, while within-ploidy treatment responses were modest, especially in 4x. Gene Ontology pointed to constitutive enrichment of proteostasis and general stress/stimulus pathways in tetraploids, consistent with a primed baseline that reduces the need for acute reprogramming. Proteomics mirrored this pattern, with 4x plants showing downshifted photosynthetic components and accumulation of chaperones and redox modules; network analysis highlighted dense folding/quality-control hubs. Salt-specific contrasts further enriched kinase and adenyl-ribonucleotide-binding functions and upregulated WRKY/MYB transcription factors linked to osmotic signaling, while immunity-related transcripts were attenuated. In the second model, potato suspension cells were exposed either to sudden polyethyleneglycol (PEG) shock or stepwise PEG adaptation. Label-free quantitative proteomics and pathway enrichment revealed that shock rapidly activated phenylpropanoid and central carbon routes while repressing translation, whereas gradual adaptation upregulated ER protein processing, mitochondrial/peroxisomal functions, and chromatin/RNA regulatory modules, with lower ROS and accumulation of selected phenolic acids, hallmarks of stabilized proteome homeostasis. In planta validation of selected genes’ expression encoding for DEPs confirmed the proteomic trend, demonstrating that the cellular adaptation model can be a powerful tool for stress-related gene discovery. Finally, functional pipelines were implemented: CRISPR/Cas knockouts and overexpression in Arabidopsis and potato, and a cisgenic promoter knock-in strategy (HDR-based with self-excision of T-DNA) to generate foreign-DNA-free, stress-tolerance edits. Collectively, the work identifies tolerance-linked gene hubs that coordinate ion/osmotic balance, redox control, and proteome stability, and delivers candidate genes and editing tools to accelerate breeding for multi-stress-resilient crops.