The activity of cysteine endopeptidases involved in the germ
The activity of cysteine endopeptidases involved in the germination of caryopses is generally regulated at two levels: the hormonal induction of synthesis by gibberellins (Drzymała et al., 2008) and a putative regulation of activity by phytocystatines (Kiyosaki et al., 2007, Martinez et al., 2009), although the latter mechanism has not been fully confirmed. There is no direct evidence that the elimination of phytocystatines leads to protease activation (Tan-Wilson and Wilson, 2012), although Hong et al. (2007) and Hwang et al. (2009) have shown that the overexpression of a gene of one phytocystatine not only reduces the activity of one of the endopeptidases during germination but also inhibits the germination process itself. Furthermore, Szewińska et al. (2013) have shown the ability of cysteine endopeptidases of the germinating triticale caryopses\' GYY 4137 morpholine salt to form in vitro complexes with a recombinant protein of one of the phytocystatines expressed in this part of the kernel.
The activation of the plant precursors of papain-like cysteine endopeptidases takes place by limited, intra- or intermolecular, proteolysis clearing of an inhibitory propeptide (Wiederanders et al., 2003), but its mechanism is not fully understood. Peptidases, which are involved in protein hydrolysis both in the developing and germinating cereal caryopses used as seed, food and fodder, are one of the factors that determine cereal quality. To date, few of these enzymes have been identified and characterised (e.g., gene sequences and molecular and kinetic protein characteristics). The EP8 endopeptidase, which is the subject of this study, is the first endopeptidase cloned from triticale. The wheat homologue of this enzyme has not been described so far. The objective of this study was to analyse the expression of EP8 in caryopses throughout development and the first hours of germination and, on the basis of correlation with the EP8 activity pattern, the formulation of assumptions regarding the regulatory mechanisms of cysteine endopeptidase activity, which is considered to be the main enzyme that degrades prolamins during germination.
Materials and methods
Results and discussion
Introduction Bacteria adapt quickly to environmental changes via acquisition of genes by horizontal gene transfer with one of three major mechanisms being conjugation (Guglielmini et al., 2011). Transmission and persistence of antibiotic resistance and virulence genes has become a worldwide health issue and resistance to several antibiotic drugs is reported for almost all pathogens, among them several Gram-positive (G+) species (Williams and Hergenrother, 2008). Conjugative plasmid transfer to recipient cells is facilitated by three major components: the relaxosome, the coupling protein and the multi-protein-complex, called a type IV secretion system (T4SS), which spans the bacterial membrane (De La Cruz et al., 2010, Fronzes et al., 2009, Wallden et al., 2010). T4SSs from Gram-negative (G-) bacteria have been studied extensively, with the Agrobacterium tumefaciens VirB/D4 system being one of the best characterized (Trokter et al., 2014). Information about T4SSs from G+ origin still remains sparse and most knowledge about those systems is based on similarity to their respective counterparts in G- bacteria (Bhatty et al., 2013, Christie et al., 2005, Guglielmini et al., 2014, Wallden et al., 2010). pIP501 is a conjugative plasmid, which was originally isolated from Streptococcus agalactiae, with the broadest transfer host-range known so far and is even able to stably exist in Escherichia coli (Kurenbach et al., 2003). This plasmid harbors multiple antibiotic resistance genes and is frequently encountered in Enterococcus faecalis and Enterococcus faecium clinical isolates (Kurenbach et al., 2003, Rosvoll et al., 2010). The pIP501 T4SS is encoded in a single operon consisting of 15 transfer genes (traA to traO) (Kurenbach et al., 2006, Wang and Macrina, 1995).