Triple activation of germinated seeds diversifies protein breakdown

Seed germination involves the metabolism of complex storage proteins, carbohydrates and other molecules into small molecules and their repurposing for new macromolecule synthesis. Here, we investigated whether activation of proteolysis could be enhanced/altered by homogenisation, bringing together a myriad of enzymes and substrates from different seed compartments, and by co-incubation of proteolytically active germinated homogenates from different species. LC/MS-MS of seeds of chickpea, lentil, mung and broccoli allowed us to track proteolytic signatures, in soaked seeds, in sprouted seeds, after homogenisation, and after co-incubation for four hours of each species, and of pairwise mixtures. The termini of peptides released at each stage showed distinct patterns of endoproteolysis, with broccoli showing a dominance of legumain-like proteolysis with Asparagine (N) directly upstream of cleavage (P1N), while the three legumes showed a more complex pattern of proteolysis with greater changes between soaked and germinated. Distinct proteolytic profiles emerged after homogenisation, with an increase in legumain-like cleavage in lentil, and an accumulation of peptides with upstream subterminal prolines (P2P) in broccoli. Likely protease-resistant N-terminally aspartate rich peptides accumulating in all species were reduced after homogenisation of broccoli (but less consistently for the legumes), suggesting that homogenisation was aiding their proteolysis. When homogenised sprouts from different germinated seeds were incubated together, we estimated the contribution of putative proteases from each individual sprout to the proteolytic profile of the other sprouts, using Ordinary Least Squares (OLS) regression. We conclude that each species’ active homogenate was shown to make a significant contribution to the N or C terminal digestion profiles of other species, indicating that mixtures of homogenised germinated seeds have the potential to release more highly digested peptides. Thus, proteolysis is activated not only by seed germination, but also by homogenisation of germinated seeds and species. Harnessing the diversity of proteolysis and other metabolic processes in triple-activated seeds shows promise as a means of improving the nutritional quality of seeds.