ARF-B₂: A Protein Complex that Specifically Binds to Part of the Anaerobic Response Element of Maize Adh 1

Abstract

Crude whole cell extracts from maize (Zea mays L.) suspension cells were examined for DNA binding proteins that specifically interact with a portion of the maize Adhl promoter that was previously shown to be in contact with a trans-acting factor in vivo. A 17 base pair, double-stranded oligonucleotide probe was constructed that centered around a strong in vivo dimethylsulfate footprint (B2) that coincides with part of the anaerobic response element (ARE). Gel retardation assays were used to characterize a major, specific DNA binding protein activity found in the crude extracts. The activity is present in both aerobic and hypoxically treated cultures and has been designated ARF-B2 (ARE binding factor). ARF-B2 appears to be a multicomponent complex, with a 54 kilodalton subunit termed ARF-B2a in primary contact with the target DNA.Maize alcohol dehydrogenase-J (Adhl) is a well-studied member of a group of genes induced as part of the plant response to hypoxic stress (6,17,18 The present report details the initial characterization of the protein:DNA interactions, specific for the ARE B2, that can be detected in extracts from maize suspension cultures in order to begin to determine the molecular mechanisms that determine physiological responses to environmental stresses. MATERIALS AND METHODS Cell CulturesMaize (Zea mays L.) cell suspension cultures (line 3377 [2], provided by J. Widholm) were maintained on commercial (GIBCO) MS medium supplemented with 2 mg/L 2,4-D. For hypoxic induction, a stream of argon was bubbled through the culture medium. Whole Cell Extracts and FractionationsWhole cell protein extracts (13) were made from the cell suspensions by the methods of McKendree et al. (14). The final dialysis buffer was NEBD with 40 mM KCI. All columns and subsequent assays were conducted in NEBD buffer, with only the noted changes in KCI concentrations.For heparin agarose chromatography, the crude extract (up to 4 mL, at 2 to 5 mg total protein/ml) was applied to a 12 mL heparin agarose (Bio-Rad) column equilibrated with NEBD at 40 mM KCI at room temperature. The column was washed with NEBD at 0.1 M KCI, then the bound proteins were eluted with NEBD at 0.6 M KCI.The 0.6 M fraction was diluted with an equal volume of NEBD without KCI (to achieve a final concentration of approximately 0.3 M KCI) and applied to a Pharmacia FPLC HR 5/5 Mono Q column that had been equilibrated with NEBD at 40 mm KC1. The column was then developed with a linear gradient of40 mM KCI to 1 M KCI in NEBD. Fractions were dialyzed against NEBD at 40 mM KCI.For analytical chromatography on FPLC Superose 6 HR 10/30, solid KCI was added to crude extract to achieve 1 M, then 200 uL of the high salt extract was applied to a Superose 6 HR 10/30 column that had been equilibrated with NEBD www.plantphysiol.org on April 4, 2019 -Published by Downloaded from

Redundant ERF-VII Transcription Factors Bind to an Evolutionarily Conserved cis-Motif to Regulate Hypoxia-Responsive Gene Expression in Arabidopsis

“…The GC-rich and GT-rich motif in the Zm ARE are distinct binding sites for the yet unidentified GCBP-1 and ARF-B(2), respectively (Ferl, 1990;Olive et al, 1991), raising the possibility that the HRPE is bound by other TFs besides ERF-VIIs. Based on single nucleotide substitutions, it is not possible to locate the exact binding site for RAP2.2 and RAP2.12 ( Figure 6).…”

“…The two interdependent subregions overlap with the promoter region that constitutively binds nuclear factors (Ferl and Nick, 1987;Ferl, 1990;Olive et al, 1991). Radiolabeled concatemers of the ARE showed decreased electrophoretic migration when incubated with nuclear extracts from maize cells.…”

Promoter elements required for developmental expression of the maize Adh1 gene in transgenic rice.

“…Two GC-and two GT-rich motifs within the ARE sequence were shown to be required for anaerobic induction in maize protoplasts (Olive et al, 1991), but the function of the ARE has not been tested in transformed plants. Specific binding of maize nuclear protein(s) to nucleotide 800 The Plant Cell sequences within the ARE has been demonstrated (Ferl, 1990; Olive et al, 1991).…”

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