We have used the maize ubiquitin 1 promoter, first exon and first intron (UBI) for rice (Oryza sativa L. cv. Taipei 309) transformation experiments and studied its expression in transgenic calli and plants. UBI directed significantly higher levels of transient gene expression than other promoter/intron combinations used for rice transformation. We exploited these high levels of expression to identify stable transformants obtained from callus-derived protoplasts co-transfected with two chimeric genes. The genes consisted of UBI fused to the coding regions of the uidA and bar marker genes (UBI:GUS and UBI:BAR). UBI:GUS expression increased in response to thermal stress in both transfected protoplasts and transgenic rice calli. Histochemical localization of GUS activity revealed that UBI was most active in rapidly dividing cells. This promoter is expressed in many, but not all, rice tissues and undergoes important changes in activity during the development of transgenic rice plants.

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The effects of gibberellic acid (GA3) and Ca2'on the synthesis and secretion of a-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, CaCt2, or both. Incubation of protoplasts in medium containing both GA3 and Ca2 , however, causes an increase in the a-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 a-amylase isozymes having high isoelectric points (pI). In the presence of GA3 alone, only isozymes with low pIs accumulate. The increase in a-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 a-amylase release. Immunoelectrophoresis shows that Ca2+ stimulates the release of low-pI aamylase isozymes by 3-fold and high-pI isozymes by 30-fold over controls incubated in GA3 alone. Immunochemical data also show that the halfmaximum concentration for this response is between 5 and 10 millimolar CaC12. The response is not specific for Ca2+ since Sr2O can substitute, although less effectively than Ca2+. Pulse-labeling experiments show that a-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 a-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.Calcium stimulates the synthesis and secretion of a-amylase in isolated barley aleurone layers that have been pretreated with GA3. Although Ca>' does not by itself induce a-amylase production, it is required, for reasons that are not understood, for maximal enzyme synthesis and secretion (5). Aleurone layers of Himalaya barley synthesize and secrete at least four isozymes of a-amylase (13). These isozymes belong to two groups that are coded on separate chromosomes (2) and are distinguished from each other by several properties including differences in their pIs2 ( 14). Aleurone layers treated with GA3 alone synthesize and secrete only one group of a-amylase isozymes, those having pIs between 4.5 and 5.2 (low-pI; 13, 14 (19). Recently it has been shown that, unlike GA3, Ca2> does not influence levels oftranslatable mRNA for high-or low-pI a-amylases (7,8). Ca2> stimulation is therefore at or between the levels of translation and release of the protein into the medium. Varner and Mense (28) proposed that Ca> facilitated a-amylase diffusion through the cell wall, but Moll and Jones (25) concluded that the mechanism for Ca" stimulation existed at the plasma membr...