Regulation of De-N-Glycosylation Enzymes in Germinating Radish Seeds

Abstract

The activities of the de-N-glycosylation enzymes endo-Kacetylp-o-glucosaminidase (ENGase; EC 3.2.1.96) and peptide44-(Kacetyl-p-D-glucosaminyl) asparagine amidase (PNCase; EC 3.5.1.52) were monitored during germination and postgerminative development in radish (Raphanus safivus L. cv Flamboyant). The ENCase activity was detected only during postgermination, whereas the PNCase activity was present at high levels in both stages. When germination was inhibited with abscisic acid or cycloheximide, PNCase activity was detected at a basic leve1 and ENCase activity was not detected at all. PNGase is present as an active protein in dry seeds and is apparently synthesized during seed formation. Conversely, the absence of ENCase in dry seeds suggests that its activity is dependent on the protein synthesis that occurs during and after germination. Treatment with gibberellic acid confirmed the production of both de-N-glycosylation enzymes after germination, and demonstrated a temporal delay between the production of the two enzymes during this period. Our results suggest that the two de-Nglycosylation enzymes are differentially regulated during plant development.The N-glycans of plant glycoproteins have been shown to play severa1 roles depending on the protein to which they are linked (Faye et al., 1989(Faye et al., , 1993. It is believed (Berger et al., 1995b) that their remova1 by means of the de-N-glycosylation enzymes ENGase (EC 3.2.1.96) and PNGase (EC 3.5.1.52) could produce changes in the function of glycoproteins. For example, de-N-glycosylation could explain the regulation of the protein activity of Con A (Bowles, 1993), which is synthesized as a glycosylated precursor (Herman et al., 1985;Chrispeels et al., 1986;Faye and Chrispeels, 1987) incapable of interacting with carbohydrate, while the active (mature) form is not a glycoprotein. In Canavalia ensformis, de-N-glycosylation appears to be routine during Con A processing in vivo (Chrispeels et al., 1986), since the seeds contain PNGase and ENGase (Sugiyama et al., 1983; Yet and Wold, 1988). In addition, the released ("free" or "unconjugated) N-glycans (UNGs), now considered to be oligosaccharins , could have a specific role in the plant cell. al., 1990) and may modulate tomato fruit ripening (Priem and Gross, 1992). The objectives of the present study were to investigate the expression of PNGase and ENGase activities in germinating radish (Xaphanus sativus L.) seeds and to examine the regulation of these enzymes during postgerminative development. MATERIALS AND METHODSSamples of commercial dry radish (Xapkanus satiuus L. cv Flamboyant) seeds (1.5 g) were placed in Petri dishes on wet filter paper and incubated for 2 h at 4°C and then at room temperature under natural light or in the dark. Three samples were used for each experimental condition. At specified times after the start of imbibition, the extent of germination was estimated by counting the number of seeds exhibiting radicle protrusion through the testa. Enzyme ExtractionEnzyme extraction was performed a...

Reboot the system thanks to protein post‐translational modifications and proteome diversity: How quiescent seeds restart their metabolism to prepare seedling establishment

“…While the biological significance of glycosylation in seed germination remains to be determined, de‐ N ‐glycosylation, i.e. the removal of N‐glycans either at the extremities or in the middle of protein sequences by specific enzymes called respectively ENGases and PNGases, was shown to occur in germinating radish and barley seeds 135, 136. These two enzymatic activities were found to occur in dry seeds with the ENGase activity being predominant in the embryo while the PNGase activity was similar in both seed compartments.…”

“…These two enzymatic activities were found to occur in dry seeds with the ENGase activity being predominant in the embryo while the PNGase activity was similar in both seed compartments. Upon imbibition, ENGase and PNGase activities increase rapidly in the embryo and endosperm, respectively 135, 136.…”

Mapping the N-linked glycosites of rice (Oryza sativa L.) germinating embryos

“…Following this, the embryo begins to synthesize a range of proteins required to support its starch metabolism, glycolysis, major carbohydrate metabolism and amino acid metabolism [ 32 – 34 ]. Like other complex physiological processes, rice seed germination is subjected to the regulation of many proteins and PTMs including N -glycosylation [ 35 , 36 ]. As yet the extent to which N- glycoproteins are of importance in the germination of the rice grain is unknown.…”