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In this study, we aimed to identify differentially accumulated proteins (DAPs) involved in PEG mock osmotic stress, cadmium (Cd 2+ ) stress, and their combined stress responses in  Brachypodium distachyon  seedling roots. The results showed that combined PEG and Cd 2+  stresses had more significant effects on  Brachypodium  seedling root growth, physiological traits, and ultrastructures when compared with each individual stress. Totally, 106 DAPs were identified that are responsive to individual and combined stresses in roots. These DAPs were mainly involved in energy metabolism, detoxification and stress defense and protein metabolism. Principal component analysis revealed that DAPs from Cd 2+  and combined stress treatments were grouped closer than those from osmotic stress treatment, indicating that Cd 2+  and combined stresses had more severe influences on the root proteome than osmotic stress alone. Protein–protein interaction analyses highlighted a 14-3-3 centered sub-network that synergistically responded to osmotic and Cd 2+  stresses and their combined stresses. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of 14 key DAP genes revealed that most genes showed consistency between transcriptional and translational expression patterns. A putative pathway of proteome metabolic changes in  Brachypodium  seedling roots under different stresses was proposed, which revealed a complicated synergetic responsive network of plant roots to adverse environments.

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Identification of differentially accumulated proteins involved in regulating independent and combined osmosis and cadmium stress response in Brachypodium seedling roots