Analysis of expression and inhibitory activity of a TrcC-6 phytocystatin present in developing and germinating seeds of triticale (×Triticosecale Wittm.)

Storage proteins of cereal seeds are processed during accumulation and degraded during germination primarily by cysteine proteinases. One of the mechanisms controlling the activity of these enzymes is the synthesis of specific inhibitors named phytocystatins. Here we present the complete gene sequence of a triticale (× Triticosecale Wittm.) phytocystatin, TrcC-6, which encodes a 152-amino acid protein with a putative 25-amino acid signal peptide. This protein has a calculated molecular mass of 16.2 kDa, and was assigned to phylogenetic group B of phytocystatins. Because TrcC-6 transcripts are present in triticale seeds, we hypothesized that this phytocystatin regulates storage protein accumulation and degradation. Therefore, changes in gene expression during the entire period of seed development and germination were examined. TrcC-6 transcripts and TrcC-6 protein levels increased during the maturation of seeds and remained high during the first hours of germination. This enabled us to conclude that TrcC-6 likely regulates seed germination by the regulation of storage protein hydrolysis. For the analysis of TrcC-6 inhibitory activity, recombinant protein was expressed in Escherichia coli BL21 (DE3) and purified. Recombinant TrcC-6 proved to be a potent inhibitor of cysteine proteinases. It inhibited the in vitro activity of papain (EC 3.4.22.2) and ficin (EC 3.4.22.3). Furthermore, native PAGE analysis revealed that recombinant TrcC-6 inhibits the activity of endogenous cysteine proteinases present in germinating seeds of triticale. Based on these results, TrcC-6 is likely one of the important factors that regulate cysteine proteinase activity during the accumulation and mobilization of storage proteins.