SummaryIn germinating rice seeds, a cysteine proteinase (REP-1), synthesized in aleurone-layer cells, is a key enzyme in the degradation of the major storage protein, glutelin. The expression of the gene for REP-1 (Rep1) is induced by gibberellins (GAs) and repressed by abscisic acid (ABA). To identify GA-responsive elements in the Rep1 promoter, we developed a transient expression system in rice aleurone cells. Deletion and pointmutation analyses indicated that the GA-response complex was composed of TAACAGA, TAACGTA, and two copies of CAACTC. The two former sequences were identical to GAREs conserved in the promoter of genes for a-amylase and proteinases in cereals. The latter, termed as CAACTC regulatory elements (CAREs), were novel GAREs. Gain-of-function experiments revealed that two pairs of GARE and CARE were necessary and sufficient to confer GA inducibility. The sequences were also required for effective transactivation by the transcription factor OsGAMyb. Four copies of either GARE or CARE showed transactivation neither by OsGAMyb nor by GA induction. CARE and GARE were also found in the promoters of a rice a-amylase gene, RAmy1A, and a barley proteinase gene, EPB1, which are expressed in germinating seeds. Mutations of CARE in their promoters caused a loss of GA inducibility and GAMyb transactivation, suggesting that CARE is the regulatory element for GA-inducible expression of hydrolase genes in the germinating seeds.

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.

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e Cellulases are enzymes that normally digest cellulose; however, some are known to play essential roles in cellulose biosynthesis. Although some endogenous cellulases of plants and cellulose-producing bacteria are reportedly involved in cellulose production, their functions in cellulose production are unknown. In this study, we demonstrated that disruption of the cellulase (carboxymethylcellulase) gene causes irregular packing of de novo-synthesized fibrils in Gluconacetobacter xylinus, a cellulose-producing bacterium. Cellulose production was remarkably reduced and small amounts of particulate material were accumulated in the culture of a cmcax-disrupted G. xylinus strain (F2-2). The particulate material was shown to contain cellulose by both solid-state 13 C nuclear magnetic resonance analysis and Fourier transform infrared spectroscopy analysis. Electron microscopy revealed that the cellulose fibrils produced by the F2-2 cells were highly twisted compared with those produced by control cells. This hypertwisting of the fibrils may reduce cellulose synthesis in the F2-2 strains.

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