Expression of alpha-amylase genes in cultured rice suspension cells is induced by sucrose starvation. To study the mechanism of sugar metabolite regulation on the expression of individual alpha-amylase genes, DNA fragments specific to each of eight rice alpha-amylase genes were synthesized and used as gene-specific probes. Comparison of the relative abundance of mRNA revealed that expression of the eight alpha-amylase genes in rice cells was differentially regulated by sucrose starvation. Accumulation of all the alpha-amylase mRNAs increased in response to sucrose starvation; however, levels of the alphaAmy3 and alphaAmy8 mRNAs were distinctly higher and constituted 90% of total alpha-amylase mRNAs. RNA gel blot and nuclear run-on transcription analyses demonstrated a positive correlation between the increased transcription rates and the elevated steady-state levels of alpha-amylase mRNAs induced by sucrose starvation. The half-lives of alphaAmy3, alphaAmy7, and alphaAmy8 were prolonged by sucrose-starvation; however, the stability of the three mRNAs seems controlled by different mechanisms. The translation inhibitors cycloheximide and anisomycin preferentially blocked the sucrose-suppressed expression of alphaAmy3 but not that of alphaAmy7 and alphaAmy8. These inhibitors also enhanced the sucrose starvation-induced accumulation of alphaAmy3 mRNA but not that of alphaAmy7 or alphaAmy8 mRNAs. Cycloheximide did not significantly alter the transcription rates of alpha-amylase genes, suggesting that labile proteins may selectively stabilize the alphaAmy7 and alphaAmy8 mRNAs but destabilize the alphaAmy3 mRNA.

In plants, sugars are required to sustain growth and regulate gene expression. A large set of genes are either up-or down-regulated by sugars; however, whether there is a common mechanism and signal transduction pathway for differential and coordinated sugar regulation remain unclear. In the present study, the rice (Oryza sativa cv Tainan 5) cell culture was used as a model system to address this question. Sucrose and glucose both played dual functions in gene regulation as exemplified by the up-regulation of growth-related genes and down-regulation of stress-related genes. Sugar coordinately but differentially activated or repressed gene expression, and nuclear run-on transcription and mRNA half-life analyses revealed regulation of both the transcription rate and mRNA stability. Although coordinately regulated by sugars, these growth-and stress-related genes were up-regulated or down-regulated through hexokinase-dependent and/or hexokinaseindependent pathways. We also found that the sugar signal transduction pathway may overlap the glycolytic pathway for gene repression. ␣-Amylase and the stress-related genes identified in this study were coordinately expressed under sugar starvation, suggesting a convergence of the nutritional and environmental stress signal transduction pathways. Together, our studies provide a new insight into the complex signal transduction network and mechanisms of sugar regulation of growth and stress-related genes in plants.

We have successfully transferred and expressed a reporter gene driven by an alpha-amylase promoter in a japonica type of rice (Oryza sativa L. cv. Tainung 62) using the Agrobacterium-mediated gene transfer system. Immature rice embryos (10-12 days after anthesis) were infected with an Agrobacterium strain carrying a plasmid containing chimeric genes of beta-glucuronidase (uidA) and neomycin phosphotransferase (nptII). Co-incubation of potato suspension culture (PSC) with the Agrobacterium inoculum significantly improved the transformation efficiency of rice. The uidA and nptII genes, which are under the control of promoters of a rice alpha-amylase gene (alpha Amy8) and Agrobacterium nopaline synthase gene (nos), respectively, were both expressed in G418-resistant calli and transgenic plants. Integration of foreign genes into the genomes of transgenic plants was confirmed by Southern blot analysis. Histochemical localization of GUS activity in one transgenic plant (R0) revealed that the rice alpha-amylase promoter functions in all cell types of the mature leaves, stems, sheaths and roots, but not in the very young leaves. This transgenic plant grew more slowly and produced less seeds than the wild-type plant, but its R1 and R2 progenies grew normally and produced as much seeds as the wild-type plant. Inheritance of foreign genes to the progenies was also confirmed by Southern blot analysis. These data demonstrate successful gene transfer and sexual inheritance of the chimeric genes.

In many plants, cysteine proteinases play essential roles in a variety of developmental and physiological processes. In rice (Oryza sativa), REP-1 is a primary cysteine proteinase responsible for the digestion of seed storage proteins to provide nutrients to support the growth of young seedlings. In the present study, the gene encoding REP-1 was isolated, characterized, and designated as OsEP3A. An OsEP3A-specific DNA probe was used to study the effect of various factors on the expression of OsEP3A in germinating seeds and vegetative tissues of rice. The expression of OsEP3A is hormonally regulated in germinating seeds, spatially and temporally regulated in vegetative tissues, and nitrogen-regulated in suspension-cultured cells. The OsEP3A promoter was linked to the coding sequence of the reporter gene, gusA, which encodes ␤-glucuronidase (GUS), and the chimeric gene was introduced into the rice genome. The OsEP3A promoter is sufficient to confer nitrogen regulation of GUS expression in suspension-cultured cells. Histochemical studies also indicate that the OsEP3A promoter is sufficient to confer the hormonal regulation of GUS expression in germinating seeds. These studies demonstrate that in rice the REP-1 protease encoded by OsEP3A may play a role in various physiological responses and processes, and that multiple mechanisms regulate the expression of OsEP3A.

No abstract