Vacuole development in cultured evacuolated oat mesophyll protoplasts

Oat leaf mesophyll protoplasts were evacuolated and shown to develop acidic vacuoles when cultured for 3 d. Vacuole development was followed by cell wall formation. Developing vacuoles, stained with acridine orange, took the form of a tubular network when viewed by confocal laser scanning microscopy. The tubules expanded and fused to form a series of interconnected vacuoles. When thin sectioned material was examined by transmission electron microscopy, the tubular network appeared as a number of small, expanding vesicles. The vacuolar H+-ATPase, H+-PPase and a membrane integral protein of 23 kDa (VM23) were shown, by Western blotting, to be removed from protoplasts following evacuolation. After 5 d culture the H+-ATPase and H+-PPase, but not VM23, were detectable in microsomal fractions. This study describes, for the first time, successful vacuole regeneration in a monocotyledenous plant. This regeneration follows a similar pattern to that seen in non-cereal protoplasts.