Mannosyl- and Xylosyl-Containing Glycans Promote Tomato (Lycopersicon esculentum Mill.) Fruit Ripening

Abstract

MATERIALS AND METHODSThe oligosacchadde glycans mannosylal-6(mannosylal-3)mannosyla1-6(mannosyla1-3)mannosyl 1-4-N-acetylglucosamine and mannosylal-6(mannosylal-3)(xylosyl81-2)mannosyl#1-4-N-acetylglucosaminyl(fucosyla1-3)N-acetylglucosamine were infiltrated into mature green tomato fruit (Lycopersicon esculentum Mill., cv Rutgers). Coinfiltration of 1 nanogram per gram fresh weight of the glycans with 40 micrograms per gram fresh weight galactose, a level of galactose insufficient to promote ripening, stimulated ripening as measured by red coloration and ethylene production.Glycoproteins contain oligosaccharide chains, i.e. glycans, linked via an N-linkage through the amido group of asparagine or an 0-linkage through the hydroxyl groups of serine, threonine, or hydroxyproline. N-Glycoproteins, i.e. N-glycosylated, have been found in plant cell walls as well as in other organelles (10). It has been suggested that glycosylation is involved with protein stability, biological activity, and mobility (1).N-Glycans have a pentasaccharidic core structure Man3(GlcNAc)2, substituted with mannosyl residues (highmannose type) or by xylosyl, fucosyl, N-acetylglucosaminyl, and galactosyl residues (complex type). Free N-glycans can occur as precursors of glycosylation or by glycoprotein proteolysis. Occurrence of the free N-glycans Man5(Xyl)-GlcNAc(Fuc)GlcNAc and Man5GlcNAc has been demonstrated in a cell suspension culture of Silene alba (9). The authors hypothesized that their presence was due to proteolysis related to autophagy. The xylosyl-containing oligosaccharide purified from the suspension culture was described as a growth factor acting at nanomolar range during early development of flax (8). We report here further evidence for the biological activity of free N-glycans in plant metabolism. The oligosaccharides Man3(Xyl)GlcNAc(Fuc)GlcNAc and Man3GlcNAc stimulated ripening oftomato fruit as measured by red coloration and ethylene production. This is the first evidence for the potential involvement of free N-glycans in fruit ripening. It is also the first description of the biological activity of the N-glycan Man5GlcNAc. Plant MaterialTomato (Lycopersicon esculentum Mill. cv Rutgers) plants were grown in a greenhouse without supplemental lighting. Flowers were pollinated by a mechanical vibrator and tagged at anthesis. Fruit of uniform size (100 ± 10 g/fresh weight) were hand-harvested at the mature green stage of ripeness at 34 ± 2 d postpollination. OligosaccharidesThe oligomannoside Man5GlcNAc was obtained commercially (BioCarb,' Lund, Sweden) and was also generously provided by Dr.

Enhancement of fruit shelf life by suppressingN-glycan processing enzymes

“…Although α-Man and β-Hex are induced by ethylene (Figs. 1H and 2H), they may also further act by promoting ethylene biosynthesis, possibly through free N-glycans, which when injected in the fruit demonstrated ethylene production (14). Altogether, our results provide evidence that α-Man and β-Hex function through degradation of cell wall glycoproteins, followed by an increase in the free N-glycans level.…”

“…Moreover, blocking of N-glycosylation with tunicamycin delays fruit ripening. Further, when injected in fruits, N-glycans are known to stimulate red coloration and ethylene production (14). To determine the N-glycan processing ability of α-Man and β-Hex, the purified enzymes were incubated with different N-glycans commonly found in fruit pericarp.…”

“…The biologic activity of free N-glycans has been noted: injection of Man 3 (Xyl)GlcNAc(Fuc)GlcNAc and Man 3 GlcNAc into mature green tomatoes stimulated ripening, as measured by the red coloration and ethylene production (14). Free N-glycans constitute a significant fraction of the soluble oligosaccharide pool in the tomato pericarp.…”

Changes in Structural Features of FreeN-Glycan and Endoglycosidase Activity during Tomato Fruit Ripening

“…Free N-glycans, which are released from glycoproteins and glycopeptides, occur ubiquitously at micromolar concentrations in various plant cells during the differentiation, growth, and ripening stages, [1][2][3][4][5][6][7][8][9][10] and auxin-like activity has been postulated. 11,12) As a part of a study to elucidate the physiological functions of free N-glycans and the significance of the de-N-glycosylation mechanism working at the differentiating and developing stages of plant cells, Kimura et al analyzed the structural features of free N-glycans occurring in developing seeds, seedlings, and shoots, [5][6][7][8][9] and the functional futures of de-N-glycosylation enzymes, endo--N-acetylglucosaminidase (ENGase) and peptide: N-glycanase (PNGase).…”

“…9,[13][14][15] They found that two types of free N-glycan, a high-mannose type and the a plant complex type, are always found in developing seeds, hypocotyls undergoing elongation, and ripening fruits. [5][6][7][8][9][10] In such free N-glycans, most high-mannose type structures have a single GlcNAc residue at their reducing end moieties, while plant complex type free N-glycans with xylose/fucose residues have an Nacetylchitobiosyl unit, indicating that the former structures are produced by ENGase and the latter structures by PNGase. It has been found that plant ENGase is highly active against high-mannose type N-glycans bearing the Man 1-2Man 1-3Man 1-4GlcNAc 1-4GlcNAc unit 9,12,13) and that plant PNGase can release high-mannose type, hybrid type, and complex type N-glycans from the peptide backbone.…”