Limited Addition of the 6-Arm ß1,2-linked N-Acetylglucosamine (GlcNAc) Residue Facilitates the Formation of the Largest N-Glycan in Plants.

The most abundant N-glycan in plants is the paucimannosidic N-glycan with core ß1,2-xylose and α1,3-fucose residues (Man3XylFuc(GlcNAc)2). Here, we report a mechanism in Arabidopsis thaliana that efficiently produces the largest N-glycan in plants. Genetic and biochemical evidence indicates that the addition of the 6-arm ß1,2-GlcNAc residue by N-acetylglucosaminyltransferase II (GnTII) is less effective than additions of the core ß1,2-xylose and α1,3-fucose residues by XylT, FucTA, and FucTB in Arabidopsis. Furthermore, analysis of gnt2 mutant and 35S:GnTII transgenic plants shows that the addition of the 6-arm non-reducing GlcNAc residue to the common N-glycan acceptor GlcNAcMan3(GlcNAc)2 inhibits additions of the core ß1,2-xylose and α1,3-fucose residues. Our findings indicate that plants limit the rate of the addition of the 6-arm GlcNAc residue to the common N-glycan acceptor as a mechanism to facilitate formation of the prevalent N-glycans with Man3XylFuc(GlcNAc)2 and (GlcNAc)2Man3XylFuc(GlcNAc)2 structures.

N-glycan containing a core α1,3-fucose residue is required for basipetal auxin transport and gravitropic response in rice (Oryza sativa).

In plants, α1,3-fucosyltransferase (FucT) catalyzes the transfer of fucose from GDP-fucose to asparagine-linked GlcNAc of the N-glycan core in the medial Golgi. To explore the physiological significance of this processing, we isolated two Oryza sativa (rice) mutants (fuct-1 and fuct-2) with loss of FucT function. Biochemical analyses of the N-glycan structure confirmed that α1,3-fucose is missing from the N-glycans of allelic fuct-1 and fuct-2. Compared with the wild-type cv Kitaake, fuct-1 displayed a larger tiller angle, shorter internode and panicle lengths, and decreased grain filling as well as an increase in chalky grains with abnormal shape. The mutant allele fuct-2 gave rise to similar developmental abnormalities, although they were milder than those of fuct-1. Restoration of a normal tiller angle in fuct-1 by complementation demonstrated that the phenotype is caused by the loss of FucT function. Both fuct-1 and fuct-2 plants exhibited reduced gravitropic responses. Expression of the genes involved in tiller and leaf angle control was also affected in the mutants. We demonstrate that reduced basipetal auxin transport and low auxin accumulation at the base of the shoot in fuct-1 account for both the reduced gravitropic response and the increased tiller angle.

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

To 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.

Auxin-Glucose Conjugation Protects the Rice (Oryza sativa L.) Seedlings Against Hydroxyurea-Induced Phytotoxicity by Activating UDP-Glucosyltransferase Enzyme.

Hydroxyurea (HU) is the replication stress known to carry out cell cycle arrest by inhibiting ribonucleotide reductase (RNR) enzyme upon generating excess hydrogen peroxide (H2O2) in plants. Phytohormones undergo synergistic and antagonistic interactions with reactive oxygen species (ROS) and redox signaling to protect plants against biotic and abiotic stress. Therefore, in this study, we investigated the protective role of Indole-3-acetic acid (IAA) in mitigating HU-induced toxicity in rice seedlings. The results showed that IAA augmentation improved the growth of the seedlings and biomass production by maintaining photosynthesis metabolism under HU stress. This was associated with reduced H2O2 and malondialdehyde (MDA) contents and improved antioxidant enzyme [superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), and peroxidase (POD)] activity that was significantly affected under HU stress. Furthermore, we showed that the HU stress-induced DNA damage leads to the activation of uridine 5'-diphosphate-glucosyltransferase (UGT), which mediates auxin homeostasis by catalyzing IAA-glucose conjugation in rice. This IAA-glucose conjugation upregulates the RNR, transcription factor 2 (E2F2), cyclin-dependent kinase (CDK), and cyclin (CYC) genes that are vital for DNA replication and cell division. As a result, perturbed IAA homeostasis significantly enhanced the key phytohormones, such as abscisic acid (ABA), salicylic acid (SA), cytokinin (CTK), and gibberellic acid (GA), that alter plant architecture by improving growth and development. Collectively, our results contribute to a better understanding of the physiological and molecular mechanisms underpinning improved growth following the HU + IAA combination, activated by phytohormone and ROS crosstalk upon hormone conjugation via UGT.