Monoclonal Antibodies; Detection Tools for Wheat Alpha‐Amylase Proteins

ABSTRACT Background and Objectives Expression of alpha‐amylase proteins during germination or other physiological events may lower grain soundness and have a negative impact on end‐use quality. The objective of the current study was to validate two new monoclonal peptide antibodies for wheat alpha‐amylases and describe alpha‐amylase expression in response to gibberellin and during germination. Findings The antibodies detected heterologously expressed alpha‐amylase protein targets and native alpha‐amylases from wheat aleurones and from germinating seeds. Conclusions The validated antibodies detected wheat alpha‐amylases expressed in the presence of gibberellin and during germination. Targeted proteomics demonstrated that alpha‐amylase expression profiles were different in the presence of gibberellin or during germination at the subgenome level. Significance and Novelty: The antibodies recognized both native and heterologously expressed alpha‐amylase proteins. Anti‐TaAMY1‐gen, developed as a generalist antibody, detected all four alpha‐amylase protein isoforms (TaAMY1‐4). Anti‐TaAMY2 was specific for TaAMY2. Furthermore, targeted proteomics allowed for identification of homeoform identity and revealed the proportion of alpha‐amylases belonging to each wheat subgenome in the presence of gibberellin and during germination. In addition, this is the first report of a simple water extraction method of alpha‐amylase proteins from crude wheatmeal that is compatible with immunoblotting.