Abstract
The promoter for the phaseolin (phas) bean seed protein gene adopts an inactive chromatin structure in leaves of transgenic tobacco. This repressive architecture, which confers stringent spatial regulation, is disrupted upon transcriptional activation during embryogenesis in a process that requires the presence of both a transcription factor (PvALF) and abscisic acid (ABA). Toward determining the need for de novo synthesis of proteins other than PvALF in transcriptional activation we explored the effect of several eukaryotic protein synthesis inhibitors. Surprisingly, cycloheximide (CHX), emetine, and verrucarin A were able to induce transcription from the phas promoter in tobacco and bean leaf tissue in the absence of either PvALF or ABA. This induction was decreased by the replication inhibitors hydroxyurea and aphidicolin but not by genistein or mimosine. Since protein phosphatases and kinases are essential components of the ABA signal transduction pathway, it is conceivable that CHX is also capable of inducing phosphorylation of proteins usually involved in ABA-mediated activation. Interestingly, okadaic acid, an inhibitor of serine/threonine phosphatase, also strongly activated transcription from the phas promoter. In contrast, the protein synthesis inhibitors anisomycin and puromycin did not activate transcription from the phas promoter, nor did the tyrosine phosphatase inhibitors phenylarsine oxide and sodium orthovanadate. These discrete but different results on transcriptional activation may reflect specific modes of action of the inhibitors, or they may reflect differential interactions of the inhibitors or of downstream events resulting from inhibitor activity with presently unknown components of the transcriptional activation system.
Highlights
The necessity of de novo protein synthesis for gene activation can be investigated through the use of inhibitors
Phas Expression Can Be Induced in Leaf Tissue by CHX—To examine whether de novo protein synthesis is required for PvALF-dependent induction of expression from the phas promoter in the presence of abscisic acid (ABA), leaves from either 58.1A or PvAlf-14 plants were subjected to CHX treatment in the presence or absence of ABA
MUG analysis showed that CHX was very effective in preventing de novo protein synthesis, since no GUS accumulation was detected in PvAlf-14 seedling leaves treated with both ABA and CHX (Fig. 1A)
Summary
ABA Induction—Seeds collected from either 58.1A plants or from line PvAlf-14 were surface-sterilized with 20% (v/v) household bleach and rinsed three times in sterile water. For fluorometric (MUG) assay, leaves or callus were homogenized in the GUS extraction buffer (50 mM NaH2PO4, pH 7.0, 10 mM EDTA, 0.1% Sarkosyl, 0.1% Triton X-100, 10 mM -mercaptoethanol) and centrifuged for 5 min in a microcentrifuge. Isolation of Histones—Approximately 5 g of leaf tissue was powdered in a mortar and pestle using liquid nitrogen, treated with nuclei isolation buffer NIB1 (0.5 M hexylene glycol, 20 mM KCl, 20 mM PIPES pH 6.5, 0.5 mM EDTA, 0.4% Triton X-100, 0.05 mM spermine, 0.125 mM spermidine, 7 mM 2-mercaptoethanol, 0.5 mM phenylmethylsulfonyl fluoride, and 0.5% (v/v) aprotonin) in the presence of protein phosphatase inhibitors (10 mM NaF, 1 mM sodium orthovanadate). The membrane containing the histones was immunochemically stained with anti-pH3 and peroxidaseconjugated goat anti-rabbit antibody (Sigma) using SuperSignal West Pico detection (Pierce)
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