Two-dimensional electrophoresis of acidic proteins isolated from ozone-stressed Norway spruce needles (Picea abies L. Karst): separation method and image processing.

The effect of ozone stress (< 10,200 or 400 micrograms m-3) on the protein pattern of Picea abies L. Karst needles was studied by fumigating potted grafts from mature trees. Clonal trees of Norway spruce were fumigated in open-top chambers for one growing season. Proteins, soluble and membrane bound, were extracted from the needles in a four-step procedure: (i) grinding in liquid nitrogen and then in a pH 5.0 buffer with thiourea added to inhibit phenol oxidase and polyvinylpolypyrrolidone (PVP) added to bind phenolic compounds; (ii) precipitation of proteins with acetone at -20 degrees C; (iii) solubilization in a modified lysis buffer; and (iv) dialysis against the modified lysis buffer. Proteins were separated by two-dimensional electrophoresis according to O'Farrell's method. The changes in the silver-stained protein pattern were evaluated by using image processing methods. Electrophoretograms from stressed and nonstressed trees are presented.