Motif mapping during chickpea germination reveals a complex sequential activation of different proteolytic activities

Despite the importance of grains and legumes in the human diet, little is known regarding peptide release and the temporal changes of protease activities during seed germination. LC/MS-MS peptidomic analysis of two cultivars of germinating chickpea followed by computational analyses indicated cleavage dominated by proteases with a single position preference (mainly before (P1) or after cleavage (P1'): L at P2 (cysEP-like); R or K at P1 (vignain-like), N or Q at P1 (legumain-like); and previously unidentified K, R, A and S at P1'; A at P2'). While P1 N cleavages were relatively constant, P1' K/R preferences were high in soaked garbanzo (kabuli) seeds, declined by four days, and returned at six days, but were much rarer in the brown (desi) cultivar. Late Embryogenesis Associated (LEA) peptides were markedly released during early germination. Vicilin peptides rich in glutamic acid near their N-termini markedly increased with germination, consistent with strong proteolytic resistance, even to human digestion, as indicated by analyses of separate datasets. Thus, this first peptidomics study of seed germination proteolytic profiles unveils a complex cultivar-specific programme of sequential activation and inactivation of a series of proteases, associated with the differential release of peptides from different protein groups.