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

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Abstract
The effects of gibberellic acid (GA3) and Ca2+ on the synthesis and secretion of α-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 GA3, CaCl2, or both. Incubation of protoplasts in medium containing both GA3 and Ca2+, however, causes an increase in the α-amylase activity of both incubation medium and tissue extract relative to controls incubated in GA3 or Ca2+ alone. Isoelectric focusing shows that adding Ca2+ to incubation media containing GA3 increases the levels of α-amylase isozymes having high isoelectric points (pI). In the presence of GA3 alone, only isozymes with low pIs accumulate. The increase in α-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 Ca2+ to maintain elevated levels of α-amylase release. Immunoelectrophoresis shows that Ca2+ stimulates the release of low-pI α-amylase isozymes by 3-fold and high-pI isozymes by 30-fold over controls incubated in GA3 alone. Immunochemical data also show that the half-maximum concentration for this response is between 5 and 10 millimolar CaCl2. The response is not specific for Ca2+ since Sr2+ can substitute, although less effectively than Ca2+. Pulse-labeling experiments show that α-amylase isozymes produced by aleurone protoplasts in response to GA3 and Ca2+ are newly synthesized. The effects of Ca2+ on the process of enzyme synthesis and secretion is not mediated via an effect of this ion on α-amylase stability or on protoplast viability. We conclude that Ca2+ directly affects the process of enzyme synthesis and transport. Experiments with protoplasts also argue against the direct involvement of the cell wall in Ca2+-stimulated enzyme release.