Studies on vacuole regeneration in evacuolated tobacco mesophyll protoplasts. Protein analysis by one‐ and two‐dimensional microgel‐electrophoresis

Abstract

Evacuolated protoplasts, P(–), have been prepared from mesophyll protoplasts. P(+), of Nicotiana tabacum L. (cv. Samsun) by centrifugation in an iso‐osmotic Percoil gradient. In comparative analyses performed with these two types of cells, vacuole proteins should appear in P(+) extracts, but be absent from freshly isolated P(–). Such differences were detectable in total protein extracts after two‐dimensional electrophoresis. Four spots were located that are likely to represent soluble vacuolar proteins. They have low molecular mass (around 20 kDa) and slightly acidic isoelectric points. In culture, evacuolated tobacco protoplasts regenerated a vacuole de novo. The vacuolation process as observed microscopically correlated welt with the reappearance of vacuolar marker enzymes. Likewise, the protein spots that were missing in the P(–)‐pattern immediately after evacuolation reappeared within the first days of protoplast culture. By immunoblotting the same behaviour was demonstrated for the well‐known vacuolar protein, tonoplast ATPase.