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

Peng-Wen Chen,Chung-An Lu,Tien-Shin Yu,Tung-Hi Tseng,Chang-Sheng Wang,Su-May Yu

doi:10.1074/jbc.m109722200

Abstract

Expression of alpha-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 alpha-amylase gene, alphaAmy3, 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 alpha-amylase gene, alphaAmy8. The alphaAmy8 SRS contains a GA response sequence and was designated as alphaAmy8 SRS/GARS. In the present study, a transgenic approach was employed to characterize the function of the alpha-amylase gene SRSs in rice. We found that the alphaAmy3 SRS significantly enhances the endogenous expression pattern of the Act1 promoter in various rice tissues throughout their developmental stages. By contrast, the alphaAmy8 SRS/GARS significantly enhances Act1 promoter activity only in embryos and endosperms of germinating rice seeds. A minimal promoter fused to the alphaAmy8 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 alphaAmy8 SRS/GARS activity. Our study demonstrates that the alpha-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.

Highlights

␣-Amylases were originally noted for their expression as regulated by GA1 and their importance in starch utilization in
The ␣Amy3 sugar response sequence (SRS) Enhances Act1 Promoter Activity in Transformed Rice Suspension Cells—Previously, we showed in a protoplast transient expression assay that insertion of three copies of the ␣Amy3 SRS at position 459 bp upstream of the transcription start site of an 846-bp Act1 promoter significantly enhanced Act1 promoter activity [11]
To determine whether similar results could be produced in stably transformed rice cells, the firefly luciferase gene (Luc) was fused downstream of the Act1 promoter or the Act1 promoter containing three tandem copies of ␣Amy3 SRS, generating Act1-Luc and Act13SRS-Luc, and the resulted chimeric genes (Fig. 2) were introduced into the rice genome via Agrobacterium-mediated transformation

Summary

Introduction

␣-Amylases were originally noted for their expression as regulated by GA1 and their importance in starch utilization in. The ␣Amy8 SRS/ GARS significantly enhances Act1 promoter activity only in embryos and endosperms of germinating rice seeds.

Results

Conclusion