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Expression and identification of intrinsically disordered proteins of in vitro sugar beet cell line during abiotic stress

Drought and heavy metal stress represent major abiotic stresses that adversely affect growth and yield of plants, changing expression of thousands of proteins. Suitable experiments on the proteomic level mostly analyze expression of globular proteins. Intrinsically disordered proteins (IDP) represent a paradigm shift in our understanding how protein structure affects functioning under physiological conditions. Due to their partially disordered structure, the proteins are involved in cell regulation, signalization and control pathways. In our study we sought to unravel the expression of IDPs during water and heavy metal stress, using model organism of an industrially important plant, sugar beet (Beta vulgaris L.) cell lines. Recently published sugar beet genome allowed us to analyze it using bioinformatics tools for a number and percentage of IDPs. DisEMBL predictor and a hotloops criterion showed that sugarbeet genome contains 41, 75% of IDPs that contain at least one long disordered region (over 30 aminoacids). Sugar beet cells were grown for two weeks on solid PG0 nutrient medium containing 300 mM mannitol (water stress) or 10, 40 or 100 µM CdCl2 (heavy metal stress). Cells were collected at 8 and 14 days. Mannitol treatment caused necrotic changes in phenotype while and a slower growth than the control cells. Only the highest used Cd concentration slowed growth of the cells and provoked necrosis, while lower concentrations improved the growth of the cells. Activities of guaiacol peroxidases, an abiotic stress biomarker, were increased after 8 and 14 days of growth of cells on mannitol and on the highest Cd concentration. Total proteins were extracted and IDPs were separated from globular proteins using native polyacrylamide gel electrophoresis (PAGE) and 8M PAGE in second dimension. Seventeen protein spots were observed after silver staining and mannitol upregulated expression of 15 proteins. Regulation of expression by Cd was concentration dependent, and the highest concentration upregulated 11 proteins. Mass spectrometry of excised protein spots identified a few confirmed IDPs but also several new ones that seem to be involved in drought and heavy metal stress.

intrinsically disordered proteins; abiotic stress; sugar beet

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