Factors affecting the re-formation of vacuoles in evacuolated protoplasts and the expression of the two vacuolar proton pumps.

The re-formation of vacuoles in miniprotoplasts (evacuolated mesophyll protoplasts) of tobacco was investigated under different conditions. When a constant osmolarity was maintained, increasing the concentration of NaCl in the medium enhanced the regeneration of vacuoles compared to the control (0.5 M mannitol used as osmoticum). An enhanced growth rate of miniprotoplasts could also be observed under low-osmolarity conditions, by substitution of NaCl for KCl or NaNO3, or with different effectors (glycinebetaine and methyljasmonate). Using the polymerase chain reaction, one cDNA fragment of the B-subunit of the vacuolar ATPase and two fragments of the tonoplast-bound pyrophosphatase (PPase) of tobacco were cloned. Southern blot analyses indicates that for both proteins more than one gene is present in tobacco. During the regeneration of vacuoles the transcript level of the PPase increased earlier than that of the B-subunit of the vacuolar ATPase under all conditions tested (0.5 M mannitol, 0.3 M mannitol, and 0.25 M NaCl, respectively). Under salt-stress conditions (0.25 M NaCl used as osmoticum), the expression level of both proton pumps is enhanced compared to the control. This increase is not specifically due to salt stress but generally to an increased growth rate of the vacuole, since under low-osmolarity conditions the expression of the vacuolar pumps is enhanced, too.