Os01g0921200
OsMOGS involves in N-glycan formation, is required for auxin-mediated root development in rice. [1]
Contents
Annotated Information
Function
To characterize further any alteration of the osmogs root system, the PRs of 5-day-old WT and osmogs seedlings were photographed using a stereomicroscope. The resulting images of adventitious root (AR) initiation, LR outgrowth and root tips, respectively, showed that the osmogs had fewer and shorter root hairs than the WT (Figure 2). Scanning electron microscopy (SEM) images also showed same result in the mature zone of osmogs root tips, compared with dense and long root hairs on the surface of the same segment of WT (Figure 2). These results confirmed that roots of osmogs were defective in root hair initiation and elongation. In plants, the newly formed oligosaccharides in the ER require further maturation in the Golgi to form high-mannose-, paucimannosidic-, hybrid- and/or complex-type N-glycans [2]. To investigate whether disruption of N-glycan processing in the ER would change overall N-glycosylation of glycoproteins, total proteins from WT and osmogs roots were immunoblotted by a lectin concanavalin A (Con A) and anti-HRP antibody. The immunoblot analysis showed a much weaker reaction with proteins from osmogs than from WT plants (Figure 6a), and suggested that formation of high-mannose, paucimannose and complex N-glycans was dramatically inhibited in osmogs. To know whether N-glycan structures were altered in osmogs, the structures of N-glycans released from glycoproteins with peptide-N-glycosidase A (PNGase A) treatment were subjected to matrix-assisted laser desorption/Ionization time-of-flight (MALDI-TOF) and electrospray ionization-fourier transform mass spectrometry (ESI-FTMS) analysis. The MALDI-TOF-MS result showed that the full MS analysis of 12 N-linked glycans contains high-mannose and complex N-glycans. The high-mannose N-glycans obviously decreased while high-mannose N-glycan with glucose residues increased in osmogs (Figure 6b and Table S3). Then three of high-mannose N-glycans and Glc3Man7GlcNAc2 from MALD-MS spectra were further quantified using ESI-FTMS analysis. From the most abundant peak areas of full ESI-FTMS, in osmogs, the three high-mannose N-glycans, Man5GlcNAc2, Man6GlcNAc2 and Man7GlcNAc2 derived from the unique MALD-MS spectra were about 1/5, 1/4 and 1/4 of that in WT respectively, while high-mannose N-glycan with glucose residues, Glc3Man7GlcNAc2 increased about 5.3 folds (Figure 6c and Table S3). Moreover, the oligosaccharide Glc3Man8GlcNAc2, which could not be detected in WT was excessively accumulated in osmogs (Table S3). These results provided solid evidence for that OsMOGS activity is required for terminal glucose trimming of N-glycan, N-glycan formation and maturation.
In order to clarify the relationship between the short-root phenotype and auxin signaling in osmogs seedlings, the auxin reporter DR5-GUS staining in 3-day-old WT and osmogs roots was observed. The staining in osmogs root tips and LRs, was much weaker than that in the WT (Figure 9a), and decreased free IAA contents in osmogs roots (Figure 9b) was consistent with the reduced DR5-GUS expression. To determine whether the lower auxin content was derived from impaired auxin transport, the rate of polar auxin transport in root tips of 3-day-old seedlings was measured. Interestingly, the IAA acropetal transport rate in the osmogs root was just half of that in the WT, and there also less inhibition by 1-naphthylphthalamic acid (NPA) on IAA acropetal transport was found in osmogs roots (Figure 9c), while basipetal IAA transport was almost not influenced in the tips of osmogs roots (Figure S5). These all indicated that the deficient auxin content was due to the decreased capacity for auxin transport in osmogs roots. To confirm whether alteration of auxin transport was involved in N-glycosylation of auxin transporters, the western blotting of two ABCB proteins, OsABCB2 and OsABCB14, was performed using OsABCB2 and OsABCB14- specific antibodies. The result clearly showed that the sizes of part of OsABCB2 and OsABCB14 decreased in osmogs roots and the smaller sizes were equal to the sizes of peptide-N-glycosidase F (PNGase F) treated OsABCB2 and OsABCB14 (Figure 9d), and suggested that involvement of OsMOGS in N-glycan processing was required for N-glycosylation of OsABCB proteins. The data demonstrated that the altered N-glycosylation of the OsABCB proteins led to an obstacle to auxin transport and abnormality of auxin signaling in osmogs roots.
Mutation
The mutant was named osmogs based on a mutation in OsMOGS (Figure 3). Compared with 7-day-old wild type (WT) plants, the osmogs plants showed retarded growth in post-embryonic roots (Figure 1a). Primary root (PR) and lateral root (LR) elongation of osmogs was inhibited dramatically and their length was approximately one-fourth and one-third of that in WT plants, respectively (Figure 1b,c). A mitotic marker reporter ProOsCYCB1;1-GUS [3] was introduced into the WT and osmogs calli. GUS staining of 5-day-old transgenic plants showed that intensity of cell division and size of the cell-dividing region had declined greatly in root tips of osmogs plants (Figure 1d), with lower cell division activity also observed in their LR primordia (Figure 1e). Longitudinal sections of the root tips of 3-day-old WT and osmogs seedlings showed root meristems of osmogs were much smaller than in the WT (Figure 1f). Moreover, cell length in the elongation zone of osmogs roots was only two-thirds of that in the WT (Figure 1g). The results clearly indicated that the shortened root phenotype of osmogs resulted from decreased cell division and elongation in the root.
Expression
To examine the OsMOGS expression pattern, the total RNA from various tissues of 4-month-old WT seedlings, including flower, panicle, flag leaf, stem, stem base and root was extracted. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) showed that OsMOGS was expressed in all tissues and organs, with its stronger expression in the root than in the shoot (Figure 4a), and suggested that roles of OsMOGS in the developing root might be more important than in the shoot. The osmogs plants displayed severe defects in the root (Figures 1a and 2), leading us to monitor dynamic OsMOGS expression in roots at early developmental stages. Total RNA from different root sections of 1–5 days after germination (DAG) in WT seedlings was isolated. The qRT-PCR result showed that OsMOGS was expressed throughout the PR, with stronger expression in the rapidly growing root and sections near the root tip than in the mature root and sections (Figure 4b), and indicated that OsMOGS was expressed predominantly in root regions where cells were undergoing rapid division and elongation. It has been predicted that the Arabidopsis GCS1/KNF possesses cytoplasmic N-terminus, ER-lumenal C-terminus and a transmembrane domain in between, but further experimental evidence is lacking [4]. Transient expression of OsMOGS–sGFP (green fluorescent protein) in leaf epidermal cells of Nicotiana benthamiana was used to determine subcellular localization of OsMOGS. The green fluorescence was found predominantly in several widespread spots on reticulum-like structures (Figure 5a). To label clearly the reticulate ER network, the OsMOGS–sGFP was co-expressed transiently with ER marker ER-rb CD3-mcherry (red fluorescence protein, RFP) [5]; the red fluorescence from RFP was also detected predominantly in punctate bodies distributed on the ER (Figure 5b). The highly overlapping GFP and RFP fluorescence signals were observed in the epidermal cells (Figure 5c), and indicated that the OsMOGS protein was localized in the ER.
Labs working on this gene
1. State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou, China 2. Key Laboratory of Crop Germplasm Resources of Zhejiang Province, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, China 3. Department of Chemistry, Changwon National University, Changwon, Gyeongnam, 641-773, Korea 4. Complex Carbohydrate Research Center, The University of Georgia, Athens, Georgia, USA 5. Department of Biochemistry and PMBBRC, Gyeongsang National University, Jinju, Korea 6. State Key Laboratory of Plant Genomics, National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China 7. State Key Laboratory of Rice Biology, China National Rice Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou, China
References
- ↑ 1.0 1.1 1.2 1.3 1.4 1.5 1.6 Wang, S., Xu, Y., Li, Z., Zhang, S., Lim, J.-M., Lee, K. O., Li, C., Qian, Q., Jiang, D. A. and Qi, Y. (2014), OsMOGS is required for N-glycan formation and auxin-mediated root development in rice (Oryza sativa L.). The Plant Journal, 78: 632–645. doi: 10.1111/tpj.12497
- ↑ Lerouge, P., Cabanes-Macheteau, M., Rayon, C., Fischette-Lainé, A.C., Gomord, V. and Faye, L. (1998) N-glycoprotein biosynthesis in plants: recent developments and future trends. Plant Mol. Biol. 38, 31–48.
- ↑ Colón-Carmona, A., You, R., Haimovitch-Gal, T. and Doerner, P. (1999) Technical advance: spatio-temporal analysis of mitotic activity with a labile cyclin-GUS fusion protein. Plant J. 20, 503–508.
- ↑ Gillmor, C.S., Poindexter, P., Lorieau, J., Palcic, M.M. and Somerville, C. (2002) α-Glucosidase I is required for cellulose biosynthesis and morphogenesis in Arabidopsis. J. Cell Biol. 156, 1003–1013.
- ↑ Nelson, B.K., Cai, X. and Nebenführ, A. (2007) A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants. Plant J. 51, 1126–1136.
