Abstract
A protein designated ABAP1 and encoded by a novel gene (GenBank accession number AF127388) was purified and shown to specifically bind abscisic acid (ABA). ABAP1 protein is a 472-amino acid polypeptide containing a WW protein interaction domain and is induced by ABA in barley aleurone layers. Polyclonal antiidiotypic antibodies (AB2) cross-reacted with purified ABAP1 and with a corresponding 52-kDa protein associated with membrane fractions of ABA-treated barley aleurones. ABAP1 genes were detected in diverse monocot and dicot species, including wheat, tobacco, alfalfa, garden pea, and oilseed rape. The recombinant ABAP1 protein optimally bound (3)H-(+)-ABA at neutral pH. Denatured ABAP1 protein did not bind (3)H-(+)-ABA, nor did bovine serum albumin. The maximum specific binding as shown by Scatchard plot analysis was 0.8 mol of ABA mol(-1) protein with a linear function of r(2) = 0.94, an indication of one ABA-binding site with a dissociation constant (K(d)) of 28 x 10(-9) m. ABA binding in aleurone plasma membranes showed a maximum binding capacity of 330 nmol of ABA g(-1) protein with a K(d) of 26.5 x 10(-9) m. The similarities in the dissociation constants for ABA binding of the recombinant protein and that of the plasma membranes suggest that the protein within the plasma membrane fraction is the native form of ABAP1. The stereospecificity of ABAP1 was established by the incapability of ABA analogs and metabolites, including (-)-ABA, trans-ABA, phaseic acid, dihydrophaseic acid, and (+)-abscisic acid-glucose ester, to displace (3)H-(+)-ABA bound to ABAP1. However, two ABA precursors, (+)-ABA aldehyde and (+)-ABA alcohol, were able to displace (3)H-(+)-ABA, an indication that the structural requirement of ABAP1 at the C-1 position is not strict. Our data show that ABAP1 exerts high binding affinity for ABA. The interaction is reversible, follows saturation kinetics, and has stereospecificity, thus meeting the criteria for an ABA-binding protein.
Highlights
A protein designated ABAP1 and encoded by a novel gene (GenBankTM accession number AF127388) was purified and shown to bind abscisic acid (ABA)
ABA Perception in Aleurones—In theory, ABA should act through a pathway that is similar to other plant hormones by binding to a receptor and triggering a signal transduction pathway leading to a cellular response
The auxin-binding proteins that are bound to plasma membrane (PM) are involved in early physiological responses, whereas those on the endoplasmic reticulum are involved in later cellular responses [9]
Summary
Chemicals—All of the chemicals were purchased from Sigma unless otherwise stated. Authentic ABA metabolites [28] were used for the stereospecificity studies and were provided by the National Research Council of Canada (Saskatoon, Saskatchewan). SDS-PAGE and Western Blot—The anti-(ϩ)-ABA monoclonal antibody (15-I-C5) (1 g), purified ABAP1 protein (0.5 and 1 g for immunoblotting), and membrane and cytosolic fractions (ϳ25 g) were loaded on a discontinuous SDS-PAGE (15% separation gel) minigel system (Bio-Rad) and separated according to the manufacturer’s instructions. After separating the digested DNA in a 0.7% agarose gel and alkaline transfer to Hybond Nϩ Nylon membranes, the blots were hybridized with the cDNA probe, ab, under the conditions described above for Northern hybridization. ABA Binding Assays—Purified ABAP1 and total PM protein were used to determine the ABA binding activity as described [15] with some modifications as follows. The incubation medium consisted of 25 mM Tris-HCl buffer, pH 7.3 (except when testing ABA binding at different pH), and 250 mM sucrose, 5 mM MgCl2, 1 mM CaCl2, 50 nM 3H-(ϩ)-ABA (except when the kinetics of ABAP1 was determined), and ABA-binding Protein. Binding was represented as the number of moles of 3H-(ϩ)-ABA/mol of ABAP1 protein for the purified ABAP1 or the number of nmol of 3H-(ϩ)-ABA/g of PM total protein
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