Serine Endopeptidase Activities of Cowpea Seeds: A Time Course during Development and Germination

Proteases in plants carry out an essential role in protein turnover during seed development and germination. Cysteine proteases (EC 3.4.22) are well-known participants of such processes. Serine proteases (EC 3.4.21), however, are far less reported as protagonists during these events. Here, we investigate this protease class acting in different cowpea seed tissues along their life cycle. In vitro enzymatic assays were performed against N-CBZ-Gly-Gly-Arg-MCA, N-CBZ-Gly-Pro-Arg- MCA, Z-Arg-Arg-MCA, and Ile-His-Pro-Phe-MCA fluorimetric substrates, under specific assay conditions. Proteins and proteases were visualized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and SDS-PAGE-gelatin, respectively. The proteolytic apparatus operating in cowpea [Vigna unguiculata (L.) Walp.] seeds includes serine endopeptidases in both cotyledons and embryo axes during the whole seed life cycle, mainly from the 12th to the 16th day after pollination and after 12 h of imbibition. The proteolytic activities appeared as bands of molecular masses between 68 and 200 kDa. Studies related to substrate specificity and pattern of inhibition showed that some activities are aprotinin-sensitive serine endopeptidases, with optimum pH ranging from 8.0 to 9.0 towards N-CBZ-GlyGly-Arg-MCA and Z-Arg-Arg-MCA, while others are phenylmethanesulphonyl fluoride (PMSF) sensitive, with an optimum pH 7.0, towards IleHis-Pro-Phe-MCA. Serine proteases acting in developing and germinating cowpea seeds are both trypsin-like (EC 3.4.21.4) and chymotrypsinlike (EC 3.4.21.1) activities.