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

Abstract

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(69His) 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(69His), 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.

Complex reproductive secretions occur in all extant gymnosperm lineages: a proteomic survey of gymnosperm pollination drops

“…Plant cystatins are protease inhibitors involved in defence against pathogens (Popovic et al 2013 ). Cystatins may also control degradation activity of plant proteases (Szewinska et al 2013 ) and regulate programmed cell death through control of associated proteases (Solomon et al 1999 ).…”

Purification and biochemical characterization of phytocystatin fromBrassica alba

“…The physiological role of phytocystatins is not well understood, although two functions have been attributed. First they play a regulatory role in enzymatic activity of endogenous cysteine proteinase, that is implicated in seed germination and maturation (Arai et al, 2002;Szewinska et al, 2013); protein turnover (Martinez et al, 2009); mobilization of storage protein, programmed cell death (Belenghi et al, 2003) and senescence (Sugawara et al, 2002). The second most important role is the defense role against attack by many viruses (Gutierrez-Campos et al, 1999), fungi (Martinez et al, 2003;Popovic et al, 2013), pests and predators, such as nematodes (Valdes-Rodriguez et al, 2007) and mites (Carrillo et al, 2011).…”

Overexpression of MpCYS2, a phytocystatin gene from Malus prunifolia (Willd.) Borkh., confers drought tolerance and protects against oxidative stress in Arabidopsis

“…Phytocystatins modulate endogenous cysteine proteases during protein turnover (Martínez et al 2009); seed maturation and germination (Arai et al 2002;Szewińska et al 2013); mobilization of storage proteins and programmed cell death (Belenghi et al 2003); and senescence (Sugawara et al 2002). They also guard against viruses (Gutierrez-Campos et al 1999), fungi Popovic et al 2013), and attacks by mites (Carrillo et al 2011) and other insects (Haq et al 2004).…”