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. Author manuscript; available in PMC: 2015 May 1.
Published in final edited form as: Plant J. 2014 Apr 15;78(4):632–645. doi: 10.1111/tpj.12497

OsMOGS is required for N-glycan formation and auxin-mediated root development in rice (Oryza sativa L.)

SuiKang Wang 1,#, YanXia Xu 1,#, ZhiLan Li 2,#, SaiNa Zhang 1, Jae-Min Lim 3,4, Kyun Oh Lee 5, ChuanYou Li 6, Qian Qian 7, De An Jiang 1, YanHua Qi 1,*
PMCID: PMC4018454  NIHMSID: NIHMS573599  PMID: 24597623

SUMMARY

N-glycosylation is a major modification of glycoproteins in eukaryotic cells. In Arabidopsis, great progress has been made in functional analysis of N-glycan production; however, there are few studies in monocotyledons. Here, we characterized a rice (Oryza sativa L.) osmogs mutant with shortened roots and isolated a gene coding a putative mannosyl-oligosaccharide glucosidase (OsMOGS), an ortholog of α-glucosidase I in Arabidopsis, which trims the terminal glucosyl residue of the oligosaccharide chain of nascent peptides in the endoplasmic reticulum (ER). OsMOGS is strongly expressed in rapidly cell-dividing tissues and OsMOGS protein is localized in the ER. Mutation of OsMOGS entirely blocked N-glycan maturation and inhibited high-mannose N-glycan formation. The osmogs mutant exhibited severe defects in root cell division and elongation, resulting in a short-root phenotype. In addition, osmogs plants had impaired root hair formation and elongation, and reduced root epidemic cell wall thickness due to decreased cellulose synthesis. Further analysis showed that auxin content and polar transport in osmogs roots were reduced due to incomplete N-glycosylation of the B subfamily of ATP-binding cassette transporter proteins (ABCBs). Our results demonstrate that involvement of OsMOGS in N-glycan formation is required for auxin-mediated root development in rice.

Keywords: OsMOGS, N-glycan formation, cellulose synthesis, auxin, root development, rice (Oryza sativa L.)

INTRODUCTION

In eukaryotic cells, N-glycosylation is an important protein co- and post-translational modification, which describes a complex process of a sugar moiety (N-glycan) being covalently linked to asparagine residues in the consensus sequence of Asn-X-Ser/Thr (where X represents any amino acid except Pro) of receptor peptides in the endoplasmic reticulum (ER) and Golgi apparatus (Golgi).

A well-conserved pathway for processing oligosaccharides in the ER has been identified in eukaryotic model organisms (Lehle et al., 2006; Song et al., 2011; Breitling and Aebi, 2013). In plants, the initial step occurs at the cytosolic side of the ER membrane, where two N-acetyglucosamines (GlcNAc) and five mannoses (Man) are added to the dolichol lipid carrier (Dol). Then this seven-sugar residue precursor is reoriented to face the ER lumen, where a further four mannoses and three glucoses (Glc) are added to form a core lipid-linked oligosaccharide Glc3Man9GlcNAc2-PP-Dol. This oligosaccharide precursor is recognized by a multi-subunit oligosaccharyltransferase (OST) complex and is transferred from the lipid to an asparagine residue of nascent peptides (Pattison and Amtmann, 2009). Subsequently, the terminal three glucosyl residues of newly synthesized glycoproteins are trimmed by α-glucosidases I and II, and four α-1,2 mannose residues are cleaved off by α-mannosidase I in the ER and Golgi (Mast and Moremen, 2006; Liebminger et al., 2009; Gomord et al., 2010). The oligosaccharides processed by the above step-by-step enzymatic catalysis are termed high-mannose N-glycans. Complex N-glycans are formed by further plant-specific coordinated stepwise processing in the Golgi (Schoberer and Strasser, 2011). N-acetylglucosaminetransferase I (GnTI) is responsible for the first step in the Golgi N-glycan processing with addition of one GlcNAc to Man5GlcNAc2 (Wenderoth and von Schaewen, 2000; Strasser et al., 2005). The enzymes involved in further pathways have been identified in plants, including Golgi alpha-mannosidase II (Strasser et al., 2006), N-acetylglucominyltransferase II (Strasser et al., 1999), β-1,2-xylosyltransferase, alpha-1,3-fucosyltransferase (FUT11/12) (Strasser et al., 2004), β-1,3-galactosyltransferase and alpha-1,4-fucosyltransferase (FUT13) (Wilson et al., 2001; Strasser et al., 2007; Qu et al., 2008).

The first N-glycan trimming step in the ER is removal of the outermost α-1,2-glucosyl residue by α-glucosidase I, and then α-glucosidase II removes the middle and innermost α-1,3-glucosyl residues. In Arabidopsis, mutation of α-glucosidase I resulted in abnormal accumulation of oligosaccharides Glc3Man7GlcNAc and Glc3Man8GlcNAc in wrinkled seeds that lacked typical protein bodies and had lower storage proteins. Interestingly, a radially swollen embryo, the most distinguishing morphology of the mutants is similar to that found with mutation in a cellulose synthase catalytic subunit CESA1/RSW1 (Boisson et al., 2001; Gillmor et al., 2002).

It is difficult to dissect the role of α-glucosidase I (GCS1) in a specific developmental stage due to embryonic lethality of the GCS1 mutation (Mayer et al., 1991; Boisson et al., 2001; Gillmor et al., 2002). When Furumizu and Komeda (2008) reported a novel hypomorphic allele of GCS1/KNOPF involved in maintaining cell shape and patterning of specialized root epidermal cells, the significance of GCS1 is delineated in post-embryonic development. Elaborate control of root cell fate and differentiation is dependent on regional auxin concentration gradients and local auxin level (Benjamins and Scheres, 2008; Overvoorde et al., 2009). Directional auxin transport and establishment of auxin gradients across tissues rely on a series of auxin transporters; of them, a B subfamily of ATP-binding cassette transporter (ABCB), previously named p-glycoprotein (PGP) has been identified as an important component of auxin transport machinery. ABCB has been recently reported to have a crucial role in auxin-mediated plant morphogenesis (Geisler and Murphy, 2006; Cho and Cho, 2012). However, the linkage between plant N-glycan production in the ER and ABCB-mediated auxin transport has not yet been established.

In the present study, we isolated a rice (Oryza sativa) mutant with severely shortened roots, which resulted from a mutation in rice mannosyl-oligosaccharide glucosidase (OsMOGS). The osmogs mutant had impaired N-glycan formation, as well as reduced cellulose and cell wall biosynthesis. We also found that osmogs roots were insensitive to exogenous auxin because of impaired polar auxin transport. Our results revealed an important role of OsMOGS in rice N-glycan formation and auxin-mediated root development.

RESULTS

Isolation and characterization of the osmogs mutant

A mutant displaying severely defective root development (Figure 1) was isolated from an ethylmethane sulfonate (EMS)-generated rice mutant library. The mutant was named osmogs based on mutation in OsMOGS (Figure 3). Compared with 7-d-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 dramatically inhibited 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 (Colón-Carmona et al., 1999) was introduced into the WT and osmogs calli. GUS staining of 5-d-old transgenic plants showed that intensity of cell division and size of the cell-dividing region had greatly declined 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-d-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 WT (Figure 1g). The results clearly indicated that the shortened root phenotype of osmogs resulted from decreased cell division and elongation in the root.

Figure 1. Phenotypic analysis of the osmogs mutant.

Figure 1

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(a) Root growth of the 7-d-old WT (left) and osmogs (right) seedlings. Bar = 2 cm.

(b) Length of primary root of the 7-d-old WT (left) and osmogs (right) seedlings.

(c) Length of lateral root of the 7-d-old WT (left) and osmogs (right) seedlings. Data in (b) and (c) represent the mean ± SD from ten independent seedlings. **P < 0.01 in Student's t-test.

(d) OsCYCB1;1-GUS activity in the root tips of the 5-d-old WT (left) and osmogs (right) seedlings. Red vertical lines indicated region of GUS staining. Bars = 200 μm.

(e) OsCYCB1;1-GUS activity in the lateral root primordia of the 5-d-old WT (left) and osmogs (right) seedlings. Red arrows indicate primordia of GUS staining. Bars = 200 μm.

(f) Longitudinal sections of the WT (left) and osmogs (right) root tips containing elongation zone of 3-d-old seedlings. Red vertical lines indicate meristematic zone. Bars = 200 μm.

(g) Cell length of elongation zone of root tips of the 3-d-old WT and osmogs seedlings. Data represent the mean ± SD from 20 independent cells. **P < 0.01 in Student's t-test.

Figure 3. Map-based cloning of OsMOGS.

Figure 3

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(a) The mutated gene locus was first mapped to a region between markers RM1387 and RM12092 in the long arm of rice chromosome 1. The fine physical map was performed by designed markers M1 and M2. The number of recombinants is marked below the individual marker, and total mapped F2 mutant population (n) is indicated on the far right.

(b) OsMOGS structure. Gray shading block, untranslated region; black shading block, exon; black line, intron.

(c) Complementation analysis of the mutant. Seven-day-old seedlings of the WT, osmogs mutant, osmogs-complemented line1 and 2 (osmogs-c1,2) (from left to right). Bars = 2 cm.

(d) Root length of the WT, osmogs mutant, osmogs-c1 and osmogs-c2. Data represent the mean ± SD from 10 independent seedlings and different letters on top of SD represent P < 0.05 in Student's t-test.

The osmogs is defective in root hair development

To further characterize alteration of the osmogs root system, the PRs of 5-d-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 to 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.

Figure 2. The osmogs mutant was defective in root hair development.

Figure 2

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Stereomicroscope images of the 5-d-old WT (left panels) and osmogs (right panels) roots, bars = 2 mm. SEM images (middle panels) of root hair elongation in the WT and osmogs primary roots marked by white boxes and arrows, bars = 200 μm.

Cloning and complementation test of OsMOGS

To analyze the osmogs genetic properties, 200 F2 progeny from a heterozygous F1 (from a cross between osmogs and O. sativa ssp. indica cv. Kasalath) were screened. The result showed that the short-root phenotype was controlled by a recessive gene (Supplemental Table 1). Map-based cloning was adopted to clone the mutated locus. First, the OsMOGS locus was mapped on a region between two simple sequence repeat (SSR) markers RM1387 and RM12092 (Figure 3a). After enlarging the population of F2 mutants (n = 1046), the OsMOGS locus was further mapped on the region between marker RM1387 and sequence-tagged site (STS) marker M1. According to the Rice Genome Annotation Project (http://rice.plantbiology.msu.edu/), there are 10 predicted genes on this region (Supplemental Table 2). When another STS marker M2 was used, a region containing only two predicted open reading frames (ORFs) was found (Figure 3a). Sequence analysis of this region from the WT and osmogs genomes demonstrated that the candidate gene was in the locus LOC_Os01g69210, which encodes a putative mannosyl-oligosaccharide glucosidase (MOGS), a GCS1 ortholog in Arabidopsis. The osmogs carried a C-to-A point mutation at 224 bp of the ORF, resulting in an Ala75 to Asp75 transition in the predicted protein (Figure 3b). According to the transmembrane prediction from TMHMM (http://www.cbs.dtu.dk/services/TMHMM/), the OsMOGS protein possesses an obvious transmembrane domain from the 62th to 85th amino acid residues, however, the property of this region is greatly altered when the 75th hydrophobic alanine changes into hydrophilic aspartic acid (Figure S1), probably resulting in the abnormal localization of the OsMOGS protein and losing its normal function in the osmogs.

According to prediction from the Conserved Domain Database (CDD) of NCBI (http://www.ncbi.nlm.nih.gov/cdd), an obvious glycohydrolase family 63 (GH63) domain was found on the C-terminus of OsMOGS. then was established. The phylogenetic tree of plant GH63 members revealed that OsMOGS was closer to other monocotyledons (e.g. Sorghum) and formed distinct clusters from dicotyledons (e.g. Arabidopsis) (Figure S2), implying that OsMOGS may play an overlapping but different role from its counterparts in rice growth and development.

To determine the correct mapping, complementation analysis was carried out by introduction of OsMOGS ORF under control of a constitutive CaMV 35S promoter into the osmogs calli. More than 10 transgenic lines were obtained and the progeny of two transformants was used for phenotypic analysis; most progeny had the same phenotype as the WT (Figure 3c,d). In addition, the short-root phenotype was also observed in the homozygous T-DNA insertion mutant lines (Figure S3). These results confirmed that the mutation in OsMOGS led to the defects in root development.

Expression pattern of OsMOGS

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. The quantitative RT-PCR (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), suggesting that roles of OsMOGS in developing root might be more important than 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 d 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), indicating that OsMOGS was predominantly expressed in root regions where cells were undergoing rapid division and elongation.

Figure 4. OsMOGS expression profile.

Figure 4

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(a) Organ-specific expression of OsMOGS. Total RNA was extracted from flower, panicle, flag leaf, stem, stem base and root of WT rice plants at mature stage.

(b) Expression of OsMOGS in different root zones of WT rice plants. Total RNA was extracted from 1, 2, 3, 4 and 5 cm sections of root tips at 1–5 d after germination (DAG) WT seedlings.

(c) Response of OsMOGS to phytohormone and stress conditions. Total RNA was extracted from roots and shoots of 7-d-old seedlings grown in normal solution and then transferred to 10 μM IAA, 1 μM 2,4-D, 1 μM NAA, 10 μM epi-BL, 10 μM ABA, 10 μM SA, 10 μM JA, 4°C and 42°C for 6 h. The OsMOGS mRNA abundance was normalized by the OsActin mRNA level. Error bars represent SD (n = 3).

To investigate the effects of various phytohormones and environmental factors on OsMOGS expression, total RNA was extracted from shoots and roots of 7-d-old WT seedlings subject to short periods of treatments, and OsMOGS expression was analyzed. OsMOGS expression was upregulated by treatments with auxin, indole-3-acetic acid (IAA), (2,4-dichlorophenyloxy)acetic acid (2,4-D) and 1-naphthylacetic acid (NAA), environmental stress (cold and heat) and stress-related phytohormones, abscisic acid (ABA), salicylic acid (SA) and jasmonic acid (JA), but not affected by cytokinin (6-benzylaminopurine, 6-BA) and brassinolide (2,4-epibrassinosteroid, EBR) (Figure 4c). It is likely that stress-related cis-elements existing in the promoter (Figure S4) conferred the ability of stress responsiveness to OsMOGS.

Subcellular localization of the OsMOGS protein

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 (Gillmor et al., 2002). Transient expression of OsMOGS–sGFP (green fluorescence 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 clearly label the reticulate ER network, the OsMOGS–sGFP was transiently co-expressed with ER marker ER-rb CD3-mcherry (red fluorescence protein, RFP) (Nelson et al., 2007); the red fluorescence from RFP was also predominantly detected 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), indicating that the OsMOGS protein was localized in the ER.

Figure 5. Subcellular localization of the OsMOGS protein.

Figure 5

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(a) and (b) Observation by two-photon laser scanning fluorescence microscopy: the epidermal cells of tobacco leaves transiently expressing the 35S promoter-driven OsMOGS–sGFP fusion protein (a) and the ER-localized marker ER-rb CD3-mcherry (b).

(c) Merged image of (a) and (b). The yellow dots with red arrows show overlap of green and red fluorescence. Bars = 100 μm.

OsMOGS is required for ER N-glycan trimming and formation

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 (Lerouge et al., 1998). 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), suggesting that formation of high-mannose, paucimannose and complex N-glycans were dramatically inhibited in osmogs.

Figure 6. N-linked glycans analysis in WT and osmogs.

Figure 6

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(a) Soluble proteins were extracted from roots of the 3-d-old WT and osmogs seedlings and separated by SDS-PAGE. Coomassie staining was used to show the equal amount of loading proteins. The lectin concanavalin A (ConA) which binds to terminal mannose residues on N-glycans and anti-horseradish peroxidase (anti-HRP) antibody which especially recognizes β1,2-xylose and core α1,3-fucose residues on N-glycans, were used to detect N-glycan types. The left and right lane of each panel indicates WT and osmogs, respectively.

(b) MALDI-TOF analysis of the structures of the N-linked glycans synthesized in WT and osmogs. The structures of oligosaccharides, including high mannose (green dotted line), complex (blue dotted line) and high mannose with glucose (red dotted line) N-glycans were indicated above their m/z peaks.

(c) The representative full MS spectra of high mannose (Man6GlcNAc2, top panels ) and high mannose with glucose (Glc3Man7GlcNAc2 bottom panels) N-linked glycans for the comparison analyses by ESI-FTMS in WT and osmogs. In (b) and (c), the blue square, green circle, blue circle, red triangle, grey star and yellow circle represents N-acetylglucosamine, mannose, glucose, fructose, xylose and galactose residue, respectively.

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 ion cyclotron resonance (ESI-FTICR) mass spectrometry (MS) analysis. The MALDI-TOF-MS result showed that the full MS analysis of twelve 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 Supplemental Table 3). Then three of high mannose N-glycans and Glc3Man7GlcNAc2 from MALD-MS spectra were further quantified using ESI-FTICR-MS analysis. From the most abundant peak areas of full ESI-FTICR-MS, 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 Supplemental Table 3). Moreover, the oligosaccharide Glc3Man8GlcNAc2 which could not be detected in WT was excessively accumulated in osmogs (Supplemental Table 3). These results provided solid evidence for that OsMOGS activity is required for terminal glucose trimming of N-glycan, N-glycan formation and maturation.

Cell wall is reduced and cellulose synthesis is influenced in osmogs

Increasing evidence has demonstrated the linkage between the N-glycan process and cellulose synthesis in plants (Lukowitz et al., 2001; Burn et al., 2002; Gillmor et al., 2002; Fanata et al., 2013). To address whether cell wall formation was affected by the blocked N-glycan process in osmogs plants, transmission electronic microscopy (TEM) images showed that cell walls of epidermal cells in the mature zone of the osmogs were much thicker (about 50%) than in WT plants (Figure 7a). The cellulose content from cell wall extract of roots was 20% less in 5-d-old osmogs seedlings than in the WT (Figure 7b). A series of reports have shown that the cellulose synthase catalytic subunit genes in rice (OsCesAs) are involved in cell wall biosynthesis (Tanaka et al., 2003; Kotake et al., 2011; Wang et al., 2012). To verify whether OsCesAs expression in osmogs seedlings was altered, total RNA was isolated from roots of 5-d-old WT and osmogs seedlings. qRT-PCR analysis showed that the mRNA abundance of six of 11 members of the OsCesA gene family (Accession numbers are listed in Table S4) in the osmogs seedlings – including OsCesA1, 5, 6, 7, 8 and 10 – was significantly lower than in the WT (Figure 7c). These data implied that cellulose synthetic activity and cell wall establishment were dependent on N-glycan formation.

Figure 7. Cell wall structure in epidermis and cellulose content in roots.

Figure 7

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(a) TEM images of epidermal cell walls in the mature zone of 5-d-old WT (left) and osmogs (right) roots. The distance of two arrows indicates cell wall thickness. Bars = 500 μm.

(b) Cellulose contents of cell walls of 5-d-old WT (left) and mutant (right) roots. Data represent the mean ± SD from six independent experiments. *P < 0.05 in Student's t-test.

(c) Expression of OsCesA gene family in 5-d-old WT (left) and osmogs (right) roots. Error bars represent SD (n=3). *P<0.05 and **P<0.01 in Student's t-test.

Auxin sensitivity of osmogs root is altered

It has been well documented that auxins not only act as endogenous developmental cues, but also mediate environmental stimuli in orchestrating root architecture (Overvoorde et al., 2010). In the present study, OsMOGS expression was affected by auxin (Figure 4c). To examine the physiological changes of the osmogs root to exogenous auxin, first, 5-d-old WT and osmogs seedlings grown on ½ Murashige and Skoog (MS) media were treated with high concentrations of auxin for 3 h respectively, and total RNA was extracted from the roots. The qRT-PCR showed that two early auxin response genes OsIAA1 (Song et al., 2009) and OsSAUR39 (Kant et al., 2009) in the osmogs roots were less induced by auxin, compared with the WT (Figure 8a). Additionally, the PR of osmogs seedlings was less inhibited compared with the WT, when the germinated WT and osmogs seedlings were grown in media containing high concentrations of IAA or 2,4-D for 7 d (Figure 8b). However, for germinated WT and osmogs seedlings which were grown in media containing different IAA concentrations for 7 d, the growth of osmogs roots was more promoted than the WT (increases in root length of 80% and 10%, respectively) when treated with lower IAA concentration (10–8 M). The osmogs root growth was still induced when the IAA concentration was so high that WT root growth was inhibited (Figure 8c). The results suggested that osmogs seedlings were less sensitive to high concentrations of auxin and that inhibition of their root growth was partly due to low concentrations of auxin.

Figure 8. Analysis of auxin response in WT and osmogs.

Figure 8

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(a) Expression of early auxin response genes OsIAA1 and OsSAUR39 in WT and osmogs roots of 5-d-old seedlings treated by 10–5 M IAA and 10–6 M 2,4-D for 3 h, respectively. The results were repeated by three independent experiments.

(b) Phenotypes of 7-d-old WT and osmogs seedlings grown in media of ½ MS (left), ½ MS + 10–5 M IAA (middle) and ½ MS + 10–7 M 2,4-D (right). Bars = 2 cm.

(c) Root growth of 7-d-old WT and osmogs mutant seedlings under gradient of the IAA treatments (from left to right: 0, 10–8, 10–7 and 10–6 M). Bars = 2 cm.

Auxin transport is impaired in osmogs roots

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-d-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-d-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), suggesting 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.

Figure 9. Analysis of auxin contents, auxin transport and glycosylation level of auxin transporters in WT and osmogs roots.

Figure 9

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(a) Auxin reporter DR5-GUS staining in the root tips and LR initiation zones of 3-d-old WT (left pane) and osmogs (right pane) seedlings. Bars = 200 μm.

(b) IAA contents in the roots of 3-d-old WT and osmogs seedlings grown in ½ MS media. FW, fresh weight. Data represent the mean ± SD from six independent experiments. **P < 0.01 in Student's t-test.

(c) IAA acropetal transport in the root tips of 3-d-old WT and osmogs seedlings grown in ½ MS media and then measured with or without 2 μM NPA treatment. Data represent the mean ± SD from ten independent experiments and different letters on top of SD represent P < 0.05 in Student's t-test.

(d) Western blotting analysis of N-glycosylation level of the OsABCB2 and OsABCB14 proteins treated with (+) and without (−) PNGase F. The arrows indicate underglycosylated proteins.

DISCUSSION

OsMOGS is required for post-embryonic root development in rice

In the present study, we cloned a rice N-glycan processing gene–OsMOGS, its GCS1/KNOPF(KNF) homolog in Arabidopsis encodes an α-glucosidase I that controls the first trimming step for N-glycans transferred from lipid to protein by the OST complex (Boisson et al., 2001). Mutations in Arabidopsis GCS1/KNF cause shrunken wrinkled seeds, cellulose deficiency and radially swollen embryos. Cell differentiation and embryo development are blocked at the late heart or early torpedo stage during embryogenesis (Boisson et al., 2001; Gillmor et al., 2002). It was not until muc/knf-101, a hypomorphic allele of GCS1/KNF affecting epidermal cell patterning in the Arabidopsis fruit and root, was isolated that the significance of α-glucosidase I in post-embryonic development in plants was shown (Furumizu and Komeda, 2008). Our results clearly demonstrated that OsMOGS controls post-embryonic root development through regulation of root cell division and elongation, which is comparable to the function of OsDGL1 (DEFECTIVE GLYCOSYLATION 1), an OST complex subunit acting upstream of OsMOGS in the ER N-glycan processing (Qin et al., 2013). Additionally, although there was ubiquitous expression of OsMOGS in mature seedlings, the strong expression in the rapidly growing region of rice radicles is an even better illustration of the crucial role of OsMOGS in root rather than in shoot development. An aggressive conclusion could be pointed out that different roles of α-glucosidase I in morphogenesis of different plant species maybe a result of different seed development profiles found in plants; the most distinguished feature of which is that the endosperm is transiently consumed by the embryo during seed development in Arabidopsis while in rice, both the embryo and endosperm are present at mature seed. Therefore, comparisons of different role of α-glucosidase I for plant viability observed in plants possessing developmentally distinct seed types, will provide further insight into the conservation, role and importance of N-glycan process in plant development.

OsMOGS is targeted to the ER and involved in N-glycan formation

Like other eukaryotic organisms, protein N-glycosylation mainly occurs in the plant ER and Golgi (Lerouge et al., 1998; Pattison and Amtmann, 2009; Schoberer and Strasser, 2011). The Arabidopsis α-1,3- and α-1,2-mannosyltransferase have been shown to be in the ER (Henquet et al., 2008; Zhang et al., 2009). Our subcellular localization experiment demonstrated that OsMOGS was targeted to the ER (Figure 5), which was consistent with its biological function in trimming the terminal glucose residue to generate the oligosaccharide Glc2Man9GlcNAc2 in the ER.

In osmogs root, mannose type and complex-type N-glycans were greatly reduced, indicating that overall efficiency of N-glycosylation of glycoproteins was affected. Similar results were also observed in Arabidopsis gcs1, rsw3 and mns1mns2mns3-1 triple-mutant seedlings with mutation in α-glucosidase I, α-glucosidase II α-subunit and class I α-mannosidases, respectively (Boisson et al., 2001; Liebminger et al., 2009; Soussilane et al., 2009), implying that N-glycan trimming reactions in the ER are essential for N-glycan formation in plants. However, the complete inhibition of complex N-glycan formation tends to be detected in the mutants with loss-of-function of Golgi-targeted N-glycan processing components (von Schaewen et al., 1993; Strasser et al., 2005, 2006; Fanata et al., 2013). It is definitely concluded that unlike mammalian cells, no alternative pathway for removal of the terminal glucosyl residue from the newly glycosylated peptide exists in plant cells; the efficient post-ER conversion to complex N-glycan is dependent on full removal of glucosyl and mannosyl residues from the new glycoprotein in the ER of plants. In addition, it is notable that the mutant lacking N-acetylglucosaminyltransferase I, complex glycan1 (cgl1), is unable to synthesize Golgi-modified complex N-glycans, but with no phenotypic changes, except for salt-sensitivity (von Schaewen et al., 1993; Kang et al., 2008). This suggests that plant growth under normal conditions is independent of complex-type N-glycans, thus the osmogs phenotypes are likely from alteration of mannose-type not complex-type N-glycans.

OsMOGS is essential to cellulose synthesis and cell wall formation in rice

Notably, mutation in the enzymes involved in the ER N-glycan processing pathways leads to cellulose deficiency during embryogenesis, although the relevant mechanism remains obscure (Lukowitz et al., 2001; Burn et al., 2002; Gillmor et al., 2002). In the present study, the cellulose content in cell walls of osmogs roots was decreased (Figure 7b), suggesting that like Arabidopsis knf mutants (Gillmor et al., 2002), lack of α-glucosidase I activity in rice also affects cellulose synthesis. As an important part of cellulose synthase complex, CesA proteins participate in synthesis of primary and secondary cell walls in higher plants (Taylor, 2008). It seems likely that reduced OsCesAs expression in the osmogs roots could at least partly contribute to cellulose deficiency in cell walls, so that the most dramatic change in the root epidermal cells observed by TEM was the modest decrease in cell wall thickness in the osmogs (Figure 7a). Our results are consistent with a recent report of the rice gnt1 mutant showing loss of N-acetylglucosaminyltransferase I (GnTI) function during the Golgi N-glycan process (Fanata et al., 2013). Clearly, cellulose synthesis in rice plants was similarly sensitive to processing defects in the ER as in the Golgi.

Another membrane-anchored gene family, endo-beta-1,4-glucanase, which is not specifically associated with CesA complexes, plays an essential role in cellulose synthesis and plant development (Mølhøj et al., 2002; Taylor, 2008). In Arabidopsis, KORRIGAN1 (KOR1) encodes one member, endo-1,4-β-D-glucanase, which acts in the cellulose assembly of the expanding cell wall and of hypocotyl and root cell elongation. Furthermore, KOR1 contains eight potential N-glycosylation sites in its extracellular domain and no true KOR1 folding, but enzymatic activity relies on N-glycosylation, whereas the types of modified N-glycans are not critical (Nicol et al., 1998; Liebminger et al., 2013). Notably, a rice osglu3 mutant with comparable root phenotype to osmogs, had a defect in a KOR1 ortholog and less crystalline cellulose content in the root cell wall (Zhang et al., 2012), providing a valuable insight into the mechanism of cellulose synthesis in an N-glycan-dependent way in higher plants.

N-glycosylation of ABCB transporters is required for auxin transport in rice

In plants, glycoproteins have been showed to affect a wide range of cellular processes, including cellulose and cell wall production, immune recognition, secret and trafficking pathways, and protein stability and folding under diverse environmental conditions (Pattison and Amtmann, 2009; Song et al., 2011). Direct measurement of IAA content and IAA polar transport showed a significantly diminished and restricted auxin distribution pattern in osmogs roots (Figure 9a–c). This result in combination with response of the osmogs root to exogenous auxin, including enhanced root growth at low concentration and decreased root inhibition at high concentration (Figure 8), led us to conclude that auxin polar transport and establishment of an auxin gradient in roots depended on N-glycosylation of glycoproteins. Increasing evidence shows that ABCB proteins act as important auxin transporters (Geisler et al., 2005; Cho et al., 2007). Analysis of N-glycosylation of OsABCB proteins suggests that reduced auxin transport in the root tip of osmogs results from underglycosylation of the OsABCB transporters, although the impact of N-glycosylation modification on the OsABCBs transport activity remains unknown.

Taken together, our data established that OsMOGS was involved in the ER N-glycan process and was required for N-glycosylation of glycoproteins, which is necessary for cellulose biosynthesis and OsABCB-mediated auxin transport in rice. Thus, further functional studies on N-glycosylation of the candidate proteins, endo-1,4-β-D-glucanases and ABCB auxin transporters will enable the unraveling of the underlying molecular mechanisms of N-glycan process control of root development in rice.

EXPERIMENTAL PROCEDURES

Plant materials and growth conditions

The mutant osmogs was isolated from an EMS-generated rice (Oryza sativa ssp. japonica cv. Dongjin) mutant library. (1) For solution culture: the seeds were soaked in 0.6–1% (v/v) HNO3 overnight and washed several times with ddH2O, then germinated at 37°C for 24 h. The germinated seeds were sown on net plates floated on culture solution (Yoshida et al., 1976). (2) For solid culture: the seeds were sterilized with 10–15% NaClO for 30 min, then washed by sterile ddH2O several times and germinated at 37°C for 24 h. The germinated seeds were placed on half-strength MS (½ MS)-agar medium. The seedlings were grown in a strictly controlled growth chamber with 30°/22°C (day/night, a photoperiod of 12 h) and 60–70% humidity.

Map-based cloning and complementation

OsMOGS was primarily mapped to the long arm of chromosome 1 with SSR markers RM165 and RM12106 using 300 F2 mutant plants. Then the locus was further mapped between RM1387 and RM12092 on a BAC clone (B1793G04, GenBank: AP006843.2). The primers M1 and M2 were designed for fine mapping, and a candidate gene selected from a 46-kb DNA region which encoded 10 putative proteins, when OsMOGS gene-specific primers were used for PCR amplification from both genomes of WT and mutant plants, and the products were sequenced. For complementation, full-length OsMOGS cDNA was cloned into a binary vector pCambia1300. The construct was introduced into callus developed from mature embryos of osmogs seeds via the Agrobacterium tumefaciens (EHA105)-mediated transformation method described by Chen et al. (2003). Primers used in this study are listed in Table S5.

RNA extraction and quantitative RT-PCR

All samples were harvested and immediately ground into powder using liquid nitrogen, and the total RNA isolated using a plant total RNA extract kit (Tiangen) according to the manufacturer's instructions. Total RNA (1 μg) was used for cDNA synthesis using a first-strand cDNA synthesis kit (TaKaRa). The real-time RT-PCR was performed as previously described (Wang et al., 2010). Primers used in this study are listed in Table S5.

Subcellular localization of OsMOGS

The full-length cDNA of OsMOGS was recombined into pENTR/SD/D-TOPO vector (Invitrogen), Then the fragment was integrated into destination vector pH7FWG2.0 under control of a constitutive CaMV 35S promoter, creating a sGFP-tagged OsMOGS fusion protein. Transient expression in epidermal cell of N. benthamiana leaves was carried out as described previously (Qi et al., 2012) and the fluorescence of fusion protein and ER marker ER-rb CD3-mcherry was detected using a confocal microscope LSM710 (Carl Zeiss, Oberkochen, Germany).

Light microscope observation and GUS staining

For observation of the whole root system, the root surface of WT and osmogs was washed with ddH2O several times, and then photographed using a Leica MZ95 stereomicroscope (Leica Instrument, Nusslosh, Germany).

The binary constructs of PromoterOsCYCB1;1-GUS and PromoterDR5-GUS were introduced into mature embryo-derived calli via Agrobacterium-mediated transformation (Hiei et al., 1994). The method of OsCYCB1;1-GUS and DR5-GUS staining was performed as described previously (Jefferson et al., 1987). After being stained, the samples were immediately rinsed in formalin acetic acid ethanol fixation solution and fixed at 4°C overnight, then observed using a LSM510 microscope (Carl Zeiss, Oberkochen, Germany).

Cell wall extract and cellulose measurement

Approximately 100 mg of fresh roots of 5-d-old WT and osmogs seedlings were ground into a fine powder using a mortar and pestle precooled with liquid nitrogen. The procedure of cell wall isolation and cellulose content determination were as previously described (Li et al., 2003; Zhang et al., 2012).

Analysis of IAA content and polar auxin transport

For IAA concentration analysis, about 20 mg of fresh roots of 3-d-old WT and osmogs seedlings grown on ½ MS-agar medium were washed by sterile deionized water several times. Then the samples were ground into a fine powder in liquid nitrogen and resolved in P-buffer [50 mM KH2PO4–NaOH with 0.02% (w/v) ascorbic acid], and 250 pg of 13C6-IAA internal standard from ProElut-C18 (http://www.dikma.com.cn) were added to each sample solution. The procedure of IAA purification and measurement was performed according to Ljung et al. (2005).

For polar auxin transport assay, 10 root segments with 1.0 cm length from root tips of 3-d-old rice seedlings were used as previously described with minor modifications (Okada et al., 1991; Dai et al., 2006; Qi et al., 2008). The radioactivity counted with a liquid scintillation counter, 1450 MicroBeta TriLux (Perkin-Elmer, Waltham, USA).

SEM and TEM

The root samples were first soaked in 2.5% glutaraldehyde solution (25% glutaraldehyde/1 M phosphate buffer (pH 7.0)/ddH2O = 1:1:8) overnight at 4°C, then fixed with 1% osmic acid for 1–2 h at room temperature. The fixed samples were dehydrated with a graded ethanol series (50%, 70%, 80%, 90%, 95% and 100% ethanol). The dehydrated samples were treated as follows: (1) for SEM, the dehydrated samples were treated with mixture of ethanol and isoamyl acetate and isoamyl acetate, respectively, then the samples were critical-point dried in liquid CO2, fastened on metallic stubs, coated with gold powder and viewed using Tabletop SEM TM-1000 (Hitachi, Tokyo, Japan); (2) for TEM, the ethanol-dehydrated samples were permeated with grated embedding agent and finally embedded in pure Spurr resin and polymerized at 70°C. The embedded samples were cut in an ultramicrotome, the thin slice was stained with 2% (w/v) uranyl acetate for 5 min and Reynolds’ lead citrate for 2 min at 25°C and then observed using a JEM-1010 EX electron microscope (JEOL, Tokyo, Japan).

N-glycorotein extract and N-linked glycan preparation

About 1.0 g of 7-d-old rice seedlings were ground in liquid nitrogen and proteins were extracted in 7.5 ml Hepes-KOH buffer (50 mM, pH 7.5) containing 20 mM sodium metabisulfite, 5 mM EDTA, 0.1% SDS, and 1.7% insoluble polyvinylpolypyrrolidone. After centrifugation, the proteins in supernatant were precipitated in pre-chilled acetone with 80% (v/v) final acetone concentration. The protein pellet was sequentially washed in pre-chilled 80% acetone and 95% ethanol. The equivalent protein samples were digested with trypsin in 100 mM Tris-HCl at pH 8.2 containing 1 mM CaCl2 at 37 °C for overnight. The tryptic digested samples were desalted by reverse phase cartridge (Sep-Pak C18, Restek). After desalting and dryness, the samples were resuspended in 50 mM sodium citrate buffer at pH 5.0 and incubated with PNGase A (Calbiochem) at 37 °C for 18 hours. The released N-linked glycans were purified by reverse phase cartridge (Sep-Pak C18) with 5% acetic acid of elution solution. The N-linked glycans were further washed with ethyl acetate 3 times for 5 mins each at room temperature. The samples were dried by lyophilization and permethylated. The permethylated glycans were further cleaned off contaminants by the reverse phase cartridge (Sep-Pak C18) and dried down for the analysis of mass spectrometry.

Analysis of N-linked glycan by mass spectrometry with MALDI-TOF and ESI-FTMS

The analysis of the permethylated N-linked glycans was performed by MALDI-TOF-MS in the reflector positive ion mode using α-dihyroxybenzoic acid (DHBA, 20 mg/mL solution in 50% methanol) as a matrix. The spectrum was obtained by using a AB SCIEX TOF/TOF 5800 MADLI mass spectrometer system. The structural and quantitative analyses of N-linked glycan were performed on the Orbitrap Fusion MS (Thermo Fisher Scientific) equipped with a Nanospray Flex Ion Source for direct infusion at 0.5 μl/min flow rate. Samples were infused onto a 30 μm fused silica emitter (New Objective). The full MS spectra were obtained for quantification with 30 s data audition time and the MS/MS spectra were obtained for structural analysis of glycans with targeted mass listed by 2 Da stepwise increases from 500 to 2000 m/z.

Protein extract and immunoblot

The EDC coupled OsABCB-specific peptides (VSRLMTRISTRMQEC for OsABCB2 and CRYLEAVLRQDVGFFDTDARTGD for OsABCB14) were used for immunizing healthy rabbits. The prepared polyclonal antibodies hybridized with total rice proteins and there were predominant bands with equal size of OsABCB proteins. Beijing Protein Institute Co. Ltd. (Beijing, China) performed the work of protein conjugation, immunization and antiserum purification as described previously (Li et al., 2011).

Protein was extracted from 0.5 g of roots of 7-d-old seedlings using a plant protein extract kit (KeyGen Biotech). (1) For N-glycans analysis, 30 μg of total protein extracts were separated by 12% SDS-PAGE gels and stained with Coomassie brilliant blue R250 or transferred to nitrocellulose membranes. The N-glycan-containing proteins were detected by peroxidase-conjugated concanavalin A (Sigma-Aldrich) and anti-horseradish peroxidase antibody (GenScript); (2) for analysis of N-glycosylation of OsABCB proteins, 40 μg of total protein extracts treated with or without PNGase F for 3 h were separated by 10% SDS-PAGE gels, then the OsABCB proteins were detected by anti-OsABCB2 and anti-OsABCB14 antibodies.

Supplementary Material

Supp FigureLegend
Supp FigureS1-S5
Supp TableS1-S5

ACKNOWLEDGMENTS

We thank Professor Akio Miyao in the Rice Genome Resource Center, Japan for providing the full-length cDNA clone of OsMOGS gene and Professor Gynheung An in the Plant Functional Genomics Laboratory, Korea for contributing T-DNA-insertional mutants of OsMOGS gene. This project was funded by grants from the National Natural Science Foundation of China (grant no. 31271692, 31171462, and 31371591), the National Science and Technology Support Plan (2012BAC09B01) and the Natural Science Foundation for Distinguished Young Scholars of Zhejiang province, China (LR13C130002). This research was also supported in part by Basic Science Research Program and the Priority Research Centers Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (grant number: NRF 2011-0013961 and NRF 2010-0029634). The mass spectrometry work produced by JML was done in the laboratory of Lance Wells, CCRC, University of Georgia and supported in part by the National Center for Biomedical Glycomics grant from NIH/INIGMS (P41GM103490).

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