Expression of alpha-amylase inhibitors in diploid Triticum species

The aim of the work was to characterize the expression of various a-amylase inhibitors (aAIs), wellknown anti-nutritional compounds, for the development of healthier diploid wheat-based functionalfoods.The salt-soluble protein fractions from the seeds of 53 accessions among Triticum monococcum subsp.monococcum (T.m.), T. monococcum subsp. boeoticum (T.b.) and Triticum urartu (T.u.) were analyzed byimmunoblotting after SDS–PAGE and Urea–PAGE using polyclonal antibodies (PABs) raised against0.19 and 0.28 aAIs expressed in bread-wheat. Reverse zymography with human saliva and Tenebrio molitora-amylases was used to assay inhibition activity. A great variability of the expression of aAI-relatedproteins was observed among T.b. and T.u. PABs, and reverse zymography revealed different bands, oftennot correlating with those present in bread-wheat. Two-dimensional electrophoresis followed by immunoblottingand mass spectrometric analysis identified these proteins as aAIs. Interestingly, no signal wasobserved within T.m. accessions. This makes T.m. an important candidate for the production of novelfunctional foods.