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The Plant Cell, Vol. 14, 2289-2301, September 2002, Copyright © 2002,
American Society of Plant Biologists

Molecular Dissection of the Gibberellin/Abscisic Acid Signaling Pathways by Transiently Expressed RNA Interference in Barley Aleurone Cells

Rodolfo Zentellaa, Daisuke Yamauchib and Tuan-hua David Ho1,a

a Plant Biology Program, Department of Biology, Washington University, St. Louis, Missouri 63130
b Department of Biological Sciences, Tokyo Metropolitan University, Tokyo 192-0397, Japan

1 To whom correspondence should be addressed. E-mail ho{at}biology.wustl.edu; fax 314-935-4432

The interaction between two phytohormones, gibberellins (GA) and abscisic acid (ABA), is an important factor regulating the developmental transition from seed dormancy to germination. In cereal aleurone tissue, GA induces and ABA suppresses the expression of {alpha}-amylases that are essential for the utilization of starch stored in the endosperm. In this work, the signaling pathways mediated by these hormones were investigated in the aleurone cells of barley seeds using double-stranded RNA interference (RNAi) technology. In this tissue, double-stranded RNA molecules generated from the transient expression of DNA templates caused a sequence-specific suppression of the target genes. We demonstrate that the transcription factor, GAMyb, is not only sufficient but also necessary for the GA induction of {alpha}-amylase. Another regulatory protein, SLN1, is shown to be a repressor of GA action, and the use of RNAi technology to inhibit the synthesis of SLN1 led to derepression of {alpha}-amylase even in the absence of GA. However, this effect still was suppressed by ABA. Although the ABA-induced Ser/Thr protein kinase, PKABA1, is known to suppress GA-induced {alpha}-amylase expression, PKABA1 RNAi did not hamper the inhibitory effect of ABA on the expression of {alpha}-amylase, indicating that a PKABA1-independent signaling pathway also may exist. We suggest that the generation of specific RNAi in a transient expression approach is a useful technique for elucidating the role of regulatory molecules in biological systems in which conventional mutational studies cannot be performed easily.




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