Globulin-1 gene expression in suspension cell cultures is dependent on exogenously supplied ABA
--Si Qing Liu, David R. Duncan and Alan L. Kriz
The most abundant protein in maize embryos is a vicilin-like storage protein encoded by the Globulin-1 (Glb1) gene. Expression of Glb1 in intact embryos is regulated by ABA, and absence of Glb1 transcripts in embryos homozygous for vp1 mutations indicates that a functional Vp1 gene product is required for Glb1 expression. These observations are consistent with the presence of consensus ABA-response sequence elements located from positions -161 to -67 (relative to the transcript start site) in the Glb1 promoter (Genetics 129:863, 1991). To further characterize the Glb1 promoter in a functional manner, we have initiated experiments designed to identify regions of the promoter which are necessary for Glb1 expression in cultured maize cells. For these experiments, a modified version of the promoter in which a novel BamHI site was introduced at position +17 to facilitate cloning was fused to GUS and a series of 5' promoter deletions were generated. Plasmid constructs were introduced into cells of the maize P3377 suspension culture by particle bombardment. Treatment of the cells with ABA (100uM) is necessary for Glb1-driven GUS expression, as no GUS activity is observed with any construct in the absence of ABA. Use of the promoter deletions in this assay indicates that sequences between -86 and -358 are necessary for Glb1 expression: high amounts of ABA-dependent expression are observed with the -358 construct, and no expression is detectable with the -86 construct, even in the presence of ABA. Experiments are in progress to further delineate the sequences required for ABA-dependent expression of the Glb1 gene. In addition, this assay system provides a rapid, sensitive means for analyzing promoter constructs in cells treated in different ways, such as varied ABA content and/or osmotic stress. We are currently collecting data from such experiments.
Another interesting observation obtained from these experiments concerns the accumulation of natural Glb1-encoded polypeptides in the P3377 cells. Immunoblot analysis of proteins extracted from these cultured cells indicates that no Glb1 proteins accumulate in the absence of ABA, as expected, and inclusion of ABA in the culture medium results in production of Glb1 protein. The protein product that accumulates, however, is the processing intermediate proproGLB1, and neither the mature GLB1 protein or its immediate precursor proGLB1 are detectable by this assay. It is also interesting to note that, in developing embryos, proproGLB1 is found exclusively in the endoplasmic reticulum, while the only Glb1 proteins found in the vacuolar fraction are proGLB1 and GLB1. These results suggest that the cellular components necessary for proper transport and processing of Glb1-encoded polypeptides are not present in the P3377 cultured cells. Analysis of Glb1 protein synthesis in these cells may be useful for studying mechanisms of globulin processing and transport.
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