The participation of phytocystatin TrcC-4 in the activity regulation of EP8, the main prolamin degrading cysteine endopeptidase in triticale seeds

Phytocystatins (PCs) are protein inhibitors of endogenous plant endopeptidases and exogenous pathogen proteinases. We have previously described the protein inhibitor TrcC-4, which is probably involved in the control of protein degradation during triticale seeds germination. The occurrence of the LARFAVXEHN motif supports the TrcC-4 designation as a PC. In this paper TrcC-4 was expressed in Escherichia coli using the pET28 expression vector. TrcC-4(6×His) was purified by affinity chromatography with a single step of purification. Western blot analysis showed the presence of TrcC-4 in both developing and germinating triticale seeds. TrcC-4 protein level was higher both in scutellum of germinating seeds and in developing grains of the triticale cultivar more resistant to pre-harvest sprouting (Zorro) than in a less resistant one (Disco). Furthermore it was demonstrated that the activity of EP8, cysteine endopeptidase responsible for the mass hydrolysis of prolamin during germination, is inhibited by TrcC-4(6×His), as confirmed by native PAGE with gliadin as a substrate. These results suggest that phytocystatin TrcC-4 controls the activity of cysteine endopeptidases involved in germination and, thus, is potentially involved in pre-harvest sprouting.