N‐glycan maturation is crucial for cytokinin‐mediated development and cellulose synthesis in Oryza sativa

Abstract

SUMMARYTo explore the physiological significance of N-glycan maturation in the plant Golgi apparatus, gnt1, a mutant with loss of N-acetylglucosaminyltransferase I (GnTI) function, was isolated in Oryza sativa. gnt1 exhibited complete inhibition of N-glycan maturation and accumulated high-mannose N-glycans. Phenotypic analyses revealed that gnt1 shows defective post-seedling development and incomplete cell wall biosynthesis, leading to symptoms such as failure in tiller formation, brittle leaves, reduced cell wall thickness, and decreased cellulose content. The developmental defects of gnt1 ultimately resulted in early lethality without transition to the reproductive stage. However, callus induced from gnt1 seeds could be maintained for periods, although it exhibited a low proliferation rate, small size, and hypersensitivity to salt stress. Shoot regeneration and dark-induced leaf senescence assays indicated that the loss of GnTI function results in reduced sensitivity to cytokinin in rice. Reduced expression of A-type O. sativa response regulators that are rapidly induced by cytokinins in gnt1 confirmed that cytokinin signaling is impaired in the mutant. These results strongly support the proposed involvement of N-glycan maturation in transport as well as in the function of membrane proteins that are synthesized via the endomembrane system.

Limited Addition of the 6-Arm β1,2-linked N-Acetylglucosamine (GlcNAc) Residue Facilitates the Formation of the Largest N-Glycan in Plants

“…Mutant plants lacking GnTI activity cannot produce hybrid and complex-type N-glycans (23)(24)(25). The addition of ␤1,2-linked GlcNAc to Man 5 (GlcNAc) 2 by GnTI is essential for the action of the subsequent N-glycan-processing enzyme Golgi ␣-mannosidase II (26 -28).…”

“…Plants without GnTI activity do not produce hybrid and complex-type N-glycans (23)(24)(25). However, when the additions of the outer ␣1,3-and ␣1,6-mannose residues to the core ␣1,6-mannose of the N-glycan precursor (dolichol lipid-linked oligosaccharide) were inhibited by crossing cgl1 with the alg3 lossof-function mutant to generate the ac double mutant, additions of the ␤1,2-xylose and ␣1,3-fucose residues to N-glycans were partially recovered (Figs.…”

Glycoengineering in plants for the development of N-glycan structures compatible with biopharmaceuticals

“…The arabidopsis mutant complex glycan 1 (cgl1), which is deficient in GnTI activity, does not show severe developmental abnormalities or reproductive disorders under normal growth conditions but exhibits growth inhibition including root swelling under salt-stress conditions (Kang et al 2008). By contrast, gnt1, a GnTI-deficient rice mutant, shows serious developmental defects under normal growth conditions and does not produce seeds (Fanata et al 2013). The GlcNAcMan 5 GlcNAc 2 N-glycan produced by GnTI is specifically used as a substrate by ManII to produce GlcNAcMan 3 GlcNAc 2 N-glycan via removal of the a1,3-mannose and a1,6-mannose residues attached at the Fig.…”

“…In accord with this, the arabidopsis mns1 mns2 double mutant produces N-glycan with Man 8 GlcNAc 2 structure as the major form but does not generate plant-specific hybrid-or complex-type N-glycans (Liebminger et al 2009). In addition, as GnTI converts the Man 5 GlcNAc 2 N-glycan structure to GlcNAcMan 5 GlcNAc 2 , mutant plants lacking GnTI activity also cannot produce hybrid-and complex-type N-glycans (Fanata et al 2013;Kang et al 2008;von Schaewen et al 1993). The arabidopsis mutant complex glycan 1 (cgl1), which is deficient in GnTI activity, does not show severe developmental abnormalities or reproductive disorders under normal growth conditions but exhibits growth inhibition including root swelling under salt-stress conditions (Kang et al 2008).…”

“…This indicates that N-glycosylation in the ER is a physiologically essential mechanism that has been well conserved during eukaryote evolution. Glycosylation mediated by enzymes present in the Golgi apparatus plays an important role in growth and development of multicellular organisms (Fanata et al 2013;Liebminger et al 2009). The Golgi apparatus represents the center of the secretory pathway as well as the place where glycosylation of glycoproteins and glycolipids is precisely regulated.…”