Ca2+-Stimulated Secretion of α-Amylase During Development in Barley Aleurone Protoplasts

The effects of gibberellic acid (GA(3)) and Ca(2+) on the synthesis and secretion of alpha-amylase from protoplasts of barley (Hordeum vulgare L. cv Himalaya) aleurone were studied. Protoplasts undergo dramatic morphological changes whether or not the incubation medium contains GA(3), CaCl(2), or both. Incubation of protoplasts in medium containing both GA(3) and Ca(2+), however, causes an increase in the alpha-amylase activity of both incubation medium and tissue extract relative to controls incubated in GA(3) or Ca(2+) alone. Isoelectric focusing shows that adding Ca(2+) to incubation media containing GA(3) increases the levels of alpha-amylase isozymes having high isoelectric points (pI). In the presence of GA(3) alone, only isozymes with low pIs accumulate. The increase in alpha-amylase activity in the incubation medium begins after 36 hours of incubation, and secretion is complete after about 72 hours. Protoplasts require continuous exposure to Ca(2+) to maintain elevated levels of alpha-amylase release. Immunoelectrophoresis shows that Ca(2+) stimulates the release of low-pI alpha-amylase isozymes by 3-fold and high-pI isozymes by 30-fold over controls incubated in GA(3) alone. Immunochemical data also show that the half-maximum concentration for this response is between 5 and 10 millimolar CaCl(2). The response is not specific for Ca(2+) since Sr(2+) can substitute, although less effectively than Ca(2+). Pulse-labeling experiments show that alpha-amylase isozymes produced by aleurone protoplasts in response to GA(3) and Ca(2+) are newly synthesized. The effects of Ca(2+) on the process of enzyme synthesis and secretion is not mediated via an effect of this ion on alpha-amylase stability or on protoplast viability. We conclude that Ca(2+) directly affects the process of enzyme synthesis and transport. Experiments with protoplasts also argue against the direct involvement of the cell wall in Ca(2+)-stimulated enzyme release.