Stored cysteine proteinases start globulin mobilization in protein bodies of embryonic axes and cotyledons during vetch ( Vicia sativa L.) seed germination

Abstract

Inhibition of protein synthesis by cycloheximide during vetch seed germination, did not prevent globulin breakdown as indicated by a decrease in vicilin- and legumin-specific immunosignals on Western blots. Protein bodies isolated from embryo axes and cotyledons of dry vetch (Vicia sativa L.) seeds using a non-aqueous method were found to be free of cytoplasmic and organellar contaminations. Lysates of these purified protein bodies were capable of degrading globulins; this process was blocked by the cysteine proteinase (CPR) inhibitor iodoacetic acid. Protein bodies contained the papain-like CPR2 and CPR4, and the legumain-like CPR VsPB2. In vitro assays showed that albumin extracts from protein bodies degraded oligopeptide substrates in the PepTag-Assay and degraded the legumain substrate N-benzoyl-asparaginyl-p-nitroanilide. We conclude that, during germination, globulin mobilization is initiated by stored CPRs in protein bodies of embryonic axes as well as cotyledons, and that de-novo-formed proteolytic enzymes mainly mediate bulk degradation of stored globulin in cotyledons after germination.

Granulosain I, a Cysteine Protease Isolated from Ripe Fruits of Solanum granuloso -leprosum (Solanaceae)

“…Protein bodies of seeds contain both storage proteins and protease precursors. The latter become activated after germination and start degradation of the stored proteins [6]. The activities of cysteine proteinases respond dramatically to different internal and external stimuli and in some cases they rise to 90% of total proteolytic activity.…”

Protein Storage Vacuoles Are Transformed into Lytic Vacuoles in Root Meristematic Cells of Germinating Seedlings by Multiple, Cell Type-Specific Mechanisms

“…These changes have been documented by following globulin mobilization using both histological stains and immunohistochemical analyses (Hö glund et al, 1992;Tiedemann et al, 2000). For example, in vetch (Vicia sativa), the first storage proteins to be mobilized, 2 to 3 d post germination, are those located in embryo axis cells; mobilization of the globulins stored in the cotyledons typically starts thereafter (Schlereth et al, 2000;Tiedemann et al, 2000). Further studies have shown that the sites of mobilization of the protein reserves coincide with the sites of growth and differentiation in the embryonic axes, such as in the radicles, where globulin breakdown starts in the epidermis and prevascular strands and then proceeds toward the embryonic shoot.…”

Expression of Sweet Potato Senescence-Associated Cysteine Proteases Affect Seed and Silique Development and Stress Tolerance in Transgenic Arabidopsis

“…Papain-like cysteine protease such as CPR2 and CPR4 are found in cotyledon and axis of dry and imbibed seeds. Gene expression studies concluded that storage globulin mobilization in germinating vetch seeds is started by the stored cysteine proteases (CPRs), however, the bulk globulin mobilization is mediated by de novo synthesized CPRs (Schlereth et al, 2000;Schlereth et al, 2001;Tiedemann et al, 2001). These data suggest that papain-like cysteine proteases may play physiological roles and functions in association with seed storage globulin protein degradation and mobilization during seed germination and seedling growth.…”

“…However, sweet potato SPAE is in close association with plant vacuolar processing enzymes of seeds from phylogenetic analysis (Chen et al, 2004). Vacuolar processing enzymes have been reported to be in association with the degradation and mobilization of globulin storage proteins during seed germination and seedling growth in Phaseolus vulgaris (Senyuk et al, 1998), Vigna mungo , Vicia sativa (Schlereth et al, 2000;Schlereth et al, 2001), and Arabidopsis thaliana (Gruis et al, 2002). In Vigna mungo, VmPE-1 has been demonstrated to increase in the cotyledons of germinating seeds and was involved in the post-translational processing of a vacuolar cysteine endopeptidase, designated SH-EP, which degraded seed storage proteins (Okamoto & www.intechopen.com Transgenic Plants -Advances and Limitations 244 Minamikawa, 1999).…”

“…There are two types of vacuoles described in plants: the storage vacuole and the lytic vacuole (Marty, 1999). Protein storage vacuoles are found in seed tissues and accumulate proteins that are re-mobilized and used as the main nutrient resource for germination (Senyuk et al, 1998;Schlereth et al, 2001). Most cells in vegetative tissues have lytic vacuoles, containing a wide range of proteases in an acidic environment.…”