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

Abstract

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 ␣ -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 ␣ -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 ␣ -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 ␣ -amylase expression, PKABA1 RNAi did not hamper the inhibitory effect of ABA on the expression of ␣ -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.

Mechanisms of Cross Talk between Gibberellin and Other Hormones

“…However, a more recent study has shown that when PKABA1 is suppressed by RNAi, ABA still inhibits the GA-induced a-amylase expression. This finding indicates that ABA affects this process through an additional, PKABA1-independent pathway (Zentella et al, 2002). A candidate alternative ABA-signaling pathway to suppress GA responses in rice may involve two ABA-induced WRKY transcriptional regulators (Xie et al, 2006).…”

Global Identification of DELLA Target Genes during Arabidopsis Flower Development

“…ABA plays an antagonistic role to GA in cereal aleurone and vegetative tissues and interacts with the GA pathway by either acting downstream of DELLA proteins (Gomez-Cadenas et al, 2001;Gubler et al, 2002;Zentella et al, 2002Zentella et al, , 2007 or stabilizing DELLA proteins (Achard et al, 2006). Several genes in response to ABA treatment were identified as RGAregulated genes in our microarray analysis.…”

The Transcription Factors HvABI5 and HvVP1 Are Required for the Abscisic Acid Induction of Gene Expression in Barley Aleurone Cells

“…The RNAi technology has been shown to cause sequence-specific repression of several target genes in barley aleurones. The activity of the endogenous factors GAMyb and SLN1, as well as the expression of transiently transformed LUC, GUS, and the Ser/ Thr protein kinase PKABA genes, were abolished using RNAi (Zentella et al, 2002). Overexpression studies cannot always reflect the in vivo function of a transcription factor.…”

“…RNAi technology has been used successfully in transient expression experiments in barley aleurone tissue (Zentella et al, 2002). To determine whether HvABI5 is necessary to induce the ABA-regulated genes HVA1 and HVA22, HvABI5 transcripts were targeted specifically and the ABA-mediated induction of ABRC-GUS reporter constructs was examined.…”