Proteolysis by CPs is crucially important for cereal seed
Proteolysis by CPs is crucially important for cereal seed development and germination. However, these enzymes must be tightly controlled. Pre-harvest sprouting is a phenomenon in which the control of CPs is disrupted. This disruption manifests in the premature synthesis and activation of CPs, leading to premature degradation of reserve proteins in the developing seed, which is not prepared to become a self-sufficient organism. Therefore, protease activities are regulated at the transcriptional level by differential gene expression and at the protein level by the activation of zymogens and by the binding of specific inhibitors and cofactors (Cambra et al., 2012).
The activation of zymogens requires proteolytic cleavage of an N-terminal pro-peptide that results in exposure of the active site of an enzyme. Cysteine proteases from the C1A family are monomeric proteins composed of two subdomains L and R with the active site in the form of a V-shaped groove between them (Novinec and Lenarcic, 2013). They are synthesized with a signal peptide 10–20 Z-IETD-FMK long and a pro-peptide (proregion) 38–250 amino acids long, followed by the sequence of the mature enzyme (Wiederanders, 2003). After synthesis, the signal peptide is removed in the lumen of the endoplasmatic reticulum and the zymogen is transported via the trans-Golgi network to senescent associated vacuoles, i.e. the lytic vacuoles, or is stored in ER-derived organelles (Santamaría et al., 2015). Zymogen is inactive because of the presence of pro-peptide that covers the active site and precludes binding of substrate but is not hydrolyzed itself due to non-productive orientation. It is considered a specific inhibitor of the enzyme, as well as an essential peptide involved in proper folding of the mature protein and in its correct subcellular targeting. An exhaustive discussion about sequences and functions of pro-peptides has been presented by Wiederanders (2003). As pro-peptides are responsible for enzyme inactivation, they provide another regulation mechanism preventing premature protease activity, also during seed germination. Barley HvPap-1 pro-peptide is a potent inhibitor of its cognate enzyme, exhibiting a tight binding inhibition against protease HvPap-1, which is involved in protein mobilization during germination of barley seeds (Cambra et al., 2012). Pro-peptides have also been shown to protect plants against pests by inhibiting their digestive CPs, as with the proregion of papaya proteinase IV, which inhibits activities of Colorado potato beetle CPs (Visal et al., 1998).
The role of phytocystatins in development and germination of cereal seeds In plants, the most direct regulators of cysteine protease activity are inhibitors belonging to the cystatin superfamily. Members of this superfamily have been divided into three families of animal cystatins (Barrett, 1987, Barrett et al., 1986) and one family of plant cystatins, the phytocystatins. The classification within families is based on sequence homology, molecular mass, and the presence of disulphide bonds and signal peptides (Turk and Bode, 1991). It was shown that cystatins interact directly with the active site clefts of the cysteine proteases through the formation of a tripartite wedge-shaped structure (Nagata et al., 2000). The three important regions of this tripartite structure are a conserved glycine residue that is located within the N-terminal region, a central segment containing the highly conserved QxVxG motif that is found in all members of this superfamily, and a tryptophan residue within the C-terminal region (Machleidt et al., 1989). In addition, phytocystatins have a conserved LARFAVDEHN-like sequence in the N-terminal region that is lacking in animal cystatins, and are characterized by the lack of disulfide bonds (Margis et al., 1998). Most phytocystatins are small proteins, with a molecular mass ranging from 11 to 16kDa, which contain only one cystatin domain (of approximately 100 residues), although some of these inhibitors contain a C-terminal extension and have molecular masses of approximately 23kDa (Martínez et al., 2005a, Shyu et al., 2004). Moreover, multidomain cystatin proteins with molecular mass of approximately 85kDa (Nissen et al., 2009, Walsh and Strickland, 1993, Wu and Haard, 2000) have also been found. A genomic analyses of peptidase inhibitors revealed that evolution of phytocystatins is probably result of extensive duplications from ancestral genes and divergence of the sequences in single clades (Santamaría et al., 2014).