International Journal
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Item 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 LeeThe 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.Item 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 LeeTo 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.Item 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 LeeIn 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.Item 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 LeeActivation 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.Item MOLECULAR CHARACTERIZATION OF PAPAYA RING SPOT VIRUS ISOLATES FROM DIFFERENT PARTS OF TAMIL NADU(2021-03-02) Nirmal Kumar, Ramasamy; V.G, Shobana; R, Kannan; P, Muthukumaran; K, AngappanAn attempt has been made to characterize different PRSV isolates which infects papaya plants were collected from different parts of Tamil Nadu. The isolates were of PRSV-P type strain and the Coat Protein gene (CP) varied in size from 840- 860 bp which encodes 280 - 284 amino acids. Sequence alignment results revealed that the seven sequences obtained from different regions shared a homology of about 90% -95% with each other and homology of about 96% with the already reported Indian isolate and 88-90% with the other country isolates collected from NCBI. KE repeats were observed in the N terminus of the CP coding region from the different isolates found out. Phylogenetic analysis revealed that the isolates identified here placed on the same group and reference isolates were grouped in a different progeny. Our study further helped us to identify conserved regions among the seven isolates and we also constructed an RNAi silencing vector targeting Coat protein gene to challenge the Papaya Ring Spot Virus (PRSV) for our future studyItem AUXIN-GLUCOSE CONJUGATION PROTECTS THE RICE (ORYZA SATIVA L.) SEEDLINGS AGAINST HYDROXYUREA-INDUCED PHYTOTOXICITY BY ACTIVATING UDP-GLUCOSYLTRANSFERASE ENZYME(2022-02-16) Vimalraj, Kantharaj; Nirmal Kumar, Ramasamy; Young-Eun, Yoon; Mi Sun, Cheong; Young-Nam, Kim; Keum-Ah, Lee; Vikranth, Kumar; Hyeonji, Choe; Song Yeob, Kim; Hadjer, Chohra; Yong Bok, LeeHydroxyurea (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.