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    LIMITED ADDITION OF THE 6-ARM Β1,2-LINKED N-ACETYLGLUCOSAMINE (GLCNAC) RESIDUE FACILITATES THE FORMATION OF THE LARGEST N-GLYCAN IN PLANTS
    (Pub Med, 2015-07-03) Jae, Yong Yoo; Ki, Seong Ko; Hyun-Kyeong, Seo; Seongha, Park; Wahyu, Indra Duwi Fanata; Rikno, Harmoko; Nirmal Kumar, Ramasamy; Thiyagarajan, Thulasinathan; Tesfaye, Mengiste; Jae-Min, Lim; Sang, Yeol Lee; Kyun, Oh Lee
    The most abundant N-glycan in plants is the paucimannosidic N-glycan with core β1,2-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 β1,2-GlcNAc residue by N-acetylglucosaminyltransferase II (GnTII) is less effective than additions of the core β1,2-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 β1,2-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.
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    N-GLYCAN MATURATION IS CRUCIAL FOR CYTOKININ-MEDIATED DEVELOPMENT AND CELLULOSE SYNTHESIS IN ORYZA SATIVA
    (Pub Med, 2013-02-25) Wahyu Indra, Duwi Fanata; Kyoung, Hwan Lee; Bo Hwa, Son; Jae, Yong Yoo; Rikno Harmoko, Harmoko; Ki, Seong Ko; Nirmal Kumar, Ramasamy; Kyung, Hwa Kim; Doo-Byoung, Oh; Hyun, Suk Jung; Jae-Yean, Kim; Sang, Yeol Lee; Kyun, Oh Lee
    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.
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    N-GLYCAN CONTAINING A CORE Α1,3-FUCOSE RESIDUE IS REQUIRED FOR BASIPETAL AUXIN TRANSPORT AND GRAVITROPIC RESPONSE IN RICE (ORYZA SATIVA)
    (2016-05-31) Rikno, Harmoko; Jae Yong, Yoo; Ki Seong, Ko; Nirmal Kumar, Ramasamy; Bo Young, Hwang; Eun, Ji Lee; Ho Soo, Kim; Kyung, Jin Lee; Doo-Byoung, Oh; Dool, -Yi Kim; Sanghun, Lee; Yang, Li; Sang Yeol, Lee; Kyun, Oh Lee
    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.
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    CELL CYCLE ARREST MEDIATED BY WEE1 IS INVOLVED IN THE UNFOLDED PROTEIN RESPONSE IN PLANTS
    (2018-09-10) Ki Seong, Ko; Jae Yong, Yoo; Nirmal Kumar, Ramasamy; Rikno, Harmoko; Bích Ngọc, Thị Vũ; Ji Ye, Park; Kyun, Oh Lee
    Activation of the unfolded protein response (UPR) in mammalian cells leads to cell cycle arrest at the G1 phase (Thomas et al., J Biol Chem 288:7606–7617, 2013). However, how UPR signaling affects cell cycle arrest remains largely unknown in plants. Here, we examined UPR and endoreduplication in Col-0, wee1, and ER stress sensing-deficient ire1a&b plants during DNA replication and ER stress conditions. We found that WEE1, an essential negative regulator of the cell cycle, is involved in the maintenance of ER homeostasis during genotoxic stress and the ER stress hypersensitivity of ire1a&b is alleviated by loss-of-function mutation in WEE1. WEE1-mediated cell cycle arrest was required for IRE1–bZIP60 pathway activation during ER stress. In contrast, loss-of-function mutation in WEE1 caused increased expression of UPR-related genes during DNA replication stress. WEE1 and IRE1 were required for endoreduplication during DNA replication stress and ER stress, respectively. Taken together, these findings suggest that cell cycle regulation is associated with UPR activation in different manners during ER stress and DNA replication stress in Arabidopsis.