Fluorescein TSA Fluorescence System Kit pathway Cystatins ar
Cystatins are potent inhibitors of cysteine proteases from the C1A family. These inhibitors primarily reduce the activities of cathepsin L-like proteases, although high concentration of barley cystatin hampers the degradation of storage proteins caused by cathepsin F-like protein (HvPap-1) (Cambra et al., 2012). Furthermore, Martínez et al. (2003a) observed that barley phytocystatin Hv-CPI inhibits several cysteine proteases, especially of bovine cathepsin-B. The inhibitory assays using chicken egg cystatin also demonstrated that both plant and animal legumain-like proteins are susceptible to cystatin inhibition (Abe et al., 1994, Chen et al., 1997). Martínez et al. (2007) showed that barley cystatin contains a C-terminal extension and can inhibit human legumain, as well as control the barley legumain-like proteinase activities in the developing endosperm. Analysis of the amino-acid sequences of PhyCys with an extended C-terminus revealed the presence of a common SNSL motif in a predicted three-dimensional structure that is similar to the backside loop of cystatins. In plants, this second domain, most likely derived from the Fluorescein TSA Fluorescence System Kit pathway of an ancestral cystatin, retains the legumain-like inhibitory properties but lacks the papain-like inhibitory domain. Therefore, C-terminal-extended PhyCys act as bifunctional protease inhibitors with the ability to control the activities of members of two cysteine protease families (Martínez et al., 2007). Protein or mRNA corresponding to phytocystatins has been identified in the seeds, fruits, leaves and roots of both monocotyledonous and dicotyledonous plants (Abe et al., 1987, Ryan et al., 1998). In seeds, phytocystatins can play three different roles, as follows: 1. regulation of protein turnover during seed maturation; 2. control of proteolysis during development and/or germination, and 3. protection of seeds against pests and pathogens. Phylogenetic classification, together with the expression patterns and the inhibitory properties of recombinant proteins against proteases shown by Abraham et al. (2006) and Martínez et al. (2009) led to the division of the Poaceae family phytocystatins into three functional groups, A, B and C, and two subgroups, C1 and C2 (Fig. 2). Members of all of the groups appear to play an important role in regulating proteolysis during the growth of cereal seeds. The best characterized phytocystatins belong to group A, and their involvement in the regulation of protein accumulation and mobilization has been documented. There is a clear correlation between the expression patterns of the genes encoding the barley cystatins in cluster A (Icy1, Icy2, Icy3, and Icy4) and their functional activities as protease inhibitors. These cystatins are preferentially expressed in dry and germinating seeds, which suggests that they are specialized endogenous regulators of the enzymes involved in mobilizing stored proteins when germination begins. A similar role has been reported for the cystatins of rice, wheat and triticale, which also cluster in group A. These proteins can inhibit CPs such as the oryzains and gliadains involved in turnover functions in the aleurone layer of rice, wheat and triticale (Arai et al., 2002, Kiyosaki et al., 2007, Szewińska et al., 2012, Szewińska et al., 2013). Rice phytocystatin OC-I probably regulates the activity of the proteases of the papain family, the expression of which was observed in dry (oryzain β) and germinating seeds (oryzain α and β), whereas OC-II inhibits oryzain γ, similar to human cathepsin H (Arai et al., 2002). The trend of increasing gene expression during the first 2 weeks of seed development and its gradual decline as the process continues have been observed for the wheat WC1–4 (Kuroda et al., 2001) and triticale TrcC-1 and TrcC-4 cystatins (Szewińska et al., 2012). Some differences in the expression profiles of these inhibitors indicate their distinct roles in seed development, maturation and germination. The expression level of the TrcC-4 gene compared with that of the TrcC-1 gene was higher during the first weeks after pollination and lasted for a much shorter period. These observations suggest that the function of TrcC-4, as was observed for WC4, might be to control of the activities of cysteine proteases, which participate in embryonic development and endosperm differentiation during the first weeks of seed development. The high level of TrcC-4 inhibitor at between 10 and 13days after pollination protects the storage proteins synthesized during the third week of seed formation. The high expression level of TrcC-4 during the first phase of development also correlates with the low expression level of EP8, the major enzyme of germinating triticale caryopses (Szewińska et al., 2013). Moreover, the pattern of EP8 transcription observed in the scutellum of a germinating caryopsis and in the developing seed is not reflected by the level of EP8 activity but is correlated with a high level of TrcC-4 protein (Fig. 3B) (Prabucka et al., 2013). Therefore, TrcC-4, which inhibits EP8 activity in vitro, might protect against the uncontrolled hydrolysis of storage proteins in germinating triticale seeds.