Proteomic changes in grape embryogenic callus in response to Agrobacterium tumefaciens-mediated transformation

Agrobacterium tumefaciens-mediated transformation is highly required for studies of grapevine gene function and of huge potential for tailored variety improvements. However, grape is recalcitrant to transformation, and the underlying mechanism is largely unknown. To better understand the overall response of grapevine to A. tumefaciens-mediated transformation, the proteomic profile of cv. Prime embryogenic callus (EC) after co-cultivation with A. tumefaciens was investigated by two-dimensional electrophoresis and MALDI-TOF-MS analysis. Over 1100 protein spots were detected in both inoculated and control EC, 69 of which showed significantly differential expression; 38 of these were successfully identified. The proteins significantly up-regulated 3 d after inoculation were PR10, resistance protein Pto, secretory peroxidase, cinnamoyl-CoA reductase and different expression regulators; down-regulated proteins were ascorbate peroxidase, tocopherol cyclase, Hsp 70 and proteins involved in the ubiquitin-associated protein-degradation pathway. A. tumefaciens transformation-induced oxidative burst and modified protein-degradation pathways were further validated with biochemical measurements. Our results reveal that agrobacterial transformation markedly inhibits the cellular ROS-removal system, mitochondrial energy metabolism and the protein-degradation machinery for misfolded proteins, while the apoptosis signaling pathway and hypersensitive response are strengthened, which might partially explain the low efficiency and severe EC necrosis in grape transformation.