Rice α-Amylase Transcriptional Enhancers Direct Multiple Mode Regulation of Promoters in Transgenic Rice

Abstract

Expression of ␣-amylase genes in cereals is induced by both gibberellin (GA) and sugar starvation. In a transient expression assay, a 105-bp sugar response sequence (SRS) in the promoter of a sugar starvation highly inducible rice ␣-amylase gene, ␣Amy3, was shown previously to confer sugar response and to enhance the activity of the rice Act1 promoter in rice protoplasts. A 230-bp SRS-like sequence was also found in the promoter of another sugar starvation highly inducible rice ␣-amylase gene, ␣Amy8. The ␣Amy8 SRS contains a GA response sequence and was designated as ␣Amy8 SRS/ GARS. In the present study, a transgenic approach was employed to characterize the function of the ␣-amylase gene SRSs in rice. We found that the ␣Amy3 SRS significantly enhances the endogenous expression pattern of the Act1 promoter in various rice tissues throughout their developmental stages. By contrast, the ␣Amy8 SRS/ GARS significantly enhances Act1 promoter activity only in embryos and endosperms of germinating rice seeds. A minimal promoter fused to the ␣Amy8 SRS/ GARS is specifically active in rice embryo and endosperm and is subject to sugar repression and GA induction in rice embryos. This sugar repression was found to override GA induction of ␣Amy8 SRS/GARS activity. Our study demonstrates that the ␣-amylase transcriptional enhancers contain cis-acting elements capable of enhancing endogenous expression patterns or activating sugar-sensitive, hormone-responsive, tissue-specific, and developmental stage-dependent expression of promoters in transgenic rice. These enhancers may facilitate the design of highly active and tightly regulated composite promoters for monocot transformation and gene expression. Our study also reveals the existence of cross-talk between the sugar and GA signaling pathways in cereals and provides a system for analyzing the underlying molecular mechanisms involved.

Interaction between Rice MYBGA and the Gibberellin Response Element Controls Tissue-Specific Sugar Sensitivity of α-Amylase Genes

“…Embryo-specific sugar repression was further demonstrated by the fusion of a 230-bp aAmy8 promoter sequence with a reporter gene and an assay in transgenic rice seeds (Chen et al, 2002). In that study, aAmy8 promoter activity was repressed by sucrose in embryos but was insensitive to sucrose repression and activated by GA in endosperms.…”

“…In rice and barley (Hordeum vulgare), the accumulation of a-amylase mRNA or protein is regulated negatively by sugars in embryos (Perata et al, 1997;Chen et al, 2002) and positively by GA in endosperms/aleurone cells Perata et al, 1997;Loreti et al, 2000). Embryo-specific sugar repression was further demonstrated by the fusion of a 230-bp aAmy8 promoter sequence with a reporter gene and an assay in transgenic rice seeds (Chen et al, 2002).…”

The SnRK1A Protein Kinase Plays a Key Role in Sugar Signaling during Germination and Seedling Growth of Rice

“…Plasmid was introduced into Agrobacterium tumefaciens strain EHA101, and rice transformation was performed as described (Chen et al, 2002). Transformed calli were cultured in a liquid MS medium, containing 3% sucrose and 10 mM 2,4-D, for the establishment of suspension cell culture as described (Yu et al, 1991).…”

Proteomic Identification of α-Amylase Isoforms Encoded byRAmy3B/3Cfrom Germinating Rice Seeds

“…11) It is now evident that the RAmy3B gene, which bears a gibberellin-response cis-element in the promoter region same as RAmy1A 12) and RAmy3E, 13) is dominantly expressed among the cluster genes at the protein level.…”

“…1 and Table 1, the expression products of RAmy1A, RAmy3B, and RAmy3E were major isoforms rice seeds germinating in 5 days. Chen et al (2002) 13) have recently demonstrated that mRNA of RAmy1A, RAmy1C, RAmy3B, and RAmy3E (Amy7, Amy10, Amy6, and Amy8) is actively expressed in the aleurone layer of 5-days germinated rice seeds. The similarity between RAmy1A and RAmy1C (Amy10) was approximately 91.5% at the N-terminal region (1 to 117), although the full protein sequence of RAmy1C is not opened yet.…”