Beijing, , China, 2 Business Unit Bioscience, Plant Research International, Wageningen University and Research Centre, 6708 PB Wageningen,

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1 The Plant Journal (2009) 60, doi: /j X x LEW3, encoding a putative a-1,2-mannosyltransferase (ALG11) in N-linked glycoprotein, plays vital roles in cell-wall biosynthesis and the abiotic stress response in Arabidopsis thaliana Min Zhang 1, Maurice Henquet 2, Zhizhong Chen 1, Hairong Zhang 1, Yi Zhang 1, Xiaozhi Ren 1, Sander van der Krol 3, Martine Gonneau 4, Dirk Bosch 2,5 and Zhizhong Gong 1,6,7,* 1 State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing, , China, 2 Business Unit Bioscience, Plant Research International, Wageningen University and Research Centre, 6708 PB Wageningen, The Netherlands, 3 Laboratory of Plant Physiology, Wageningen University, The Netherlands, 4 Laboratoire de Biologie Cellulaire, Unité de Recherche 501, Institut Jean-Pierre Bourgin Institut National de la Recherche Agronomique, Route de St Cyr, Versailles Cedex, France, 5 Membrane Enzymology, Department of Chemistry, Utrecht University, 3584 CH Utrecht, The Netherlands, 6 China Agricultural University/University of California Riverside Center for Biological Sciences and Biotechnology, and 7 National Center for Plant Gene Research, Beijing , China Received 4 June 2009; revised 19 August 2009; accepted 27 August 2009; published online 22 September * For correspondence (fax ; gongzz@cau.edu.cn). SUMMARY N-linked glycosylation is an essential protein modification that helps protein folding, trafficking and translocation in eukaryotic systems. The initial process for N-linked glycosylation shares a common pathway with assembly of a dolichol-linked core oligosaccharide. Here we characterize a new Arabidopsis thaliana mutant lew3 (leaf wilting 3), which has a defect in an a-1,2-mannosyltransferase, a homolog of ALG11 in yeast, that transfers mannose to the dolichol-linked core oligosaccharide in the last two steps on the cytosolic face of the ER in N-glycan precursor synthesis. LEW3 is localized to the ER membrane and expressed throughout the plant. Mutation of LEW3 caused low-level accumulation of Man 3 GlcNAc 2 and Man 4 GlcNAc 2 glycans, structures that are seldom detected in wild-type plants. In addition, the lew3 mutant has low levels of normal highmannose-type glycans, but increased levels of complex-type glycans. The lew3 mutant showed abnormal developmental phenotypes, reduced fertility, impaired cellulose synthesis, abnormal primary cell walls, and xylem collapse due to disturbance of the secondary cell walls. lew3 mutants were more sensitive to osmotic stress and abscisic acid (ABA) treatment. Protein N-glycosylation was reduced and the unfolded protein response was more activated by osmotic stress and ABA treatment in the lew3 mutant than in the wild-type. These results demonstrate that protein N-glycosylation plays crucial roles in plant development and the response to abiotic stresses. Keywords: protein N-glycosylation, a-1,2-mannosyltransferase, unfolded protein response, abiotic stresses. INTRODUCTION In eukaryotic cells, N-linked glycosylation is an essential protein modification and shares a common pathway with the synthesis of core dolichol-linked oligosaccharides in the endoplasmic reticulum (Figure 1) (Herscovics and Orlean, 1993; Burda and Aebi, 1999; Gemmill and Trimble, 1999; Cipollo et al., 2001; Helenius and Aebi, 2004; O Reilly et al., 2006; Weerapana and Imperiali, 2006). Most genes involved in this pathway have been identified in yeast. First, on the cytosolic surface of the ER, a series of glycotransferases add two N-acetylglucosamines (GlcNAc) and five mannoses stepwise to a lipid carrier, dolichylphosphate (Herscovics and Orlean, 1993; Burda and Aebi, 1999; Gemmill and 983 Journal compilation ª 2009 Blackwell Publishing Ltd

2 984 Min Zhang et al. Figure 1. N-glycosylation in Arabidopsis. Wild type pathway: In wild-type plants, N-glycosylation starts with assembly of the oligosaccharide Man 5 GlcNAc 2 to the lipid carrier dolichol phosphate (PPdolichol), then the glycan flips to the luminal side of ER and is further extended by glycosyltransferases. Subsequently, the lipid-bound oligosaccharide is transferred by the OST complex onto selected Asn residues of nascent secretory polypeptides, and modified to form the complex glycan structure. Proposed lew3 mutant pathway: Absence of LEW3 activity results in Man 3 GlcNAc 2 lipid-bound precursors that can be flipped across the ER membrane. This structure can only be partially extended by mannose residues, and these aberrant glycans are transferred to glycoproteins by the OST complex. Subsequently, these aberrant glycans may be further processed into regular complex-type plant N-glycans by glycosidases and glycosyltransferases in the ER and Golgi. Both pathways are operational in the leaky lew3 mutant. The enzymes involved in N-glycosylation are labeled by letters: a, ALG (asparagine-linked glycosylation) enzymes; b, OST subunits (DAD1, STT3, DGL1); c, glucosidases I and II; d, mannosidase I; e, GnTI (N-acetylglucosaminyltransferase I); LEW3/ALG11, Dol-P- Man:Man 3,4 GlcNAc 2 -PP-Dol a-1,2-mannosyltransferase. Trimble, 1999; Weerapana and Imperiali, 2006). ALG11, an a-1,2-mannosyltransferase, catalyzes the last two steps of assembly of the oligosaccharide Man 5 GlcNAc 2 -PP-dolichol, before it is flipped to the lumen of the ER (Cipollo et al., 2001; O Reilly et al., 2006). Then, the seven sugars from dolichol- P-Man- and dolichol-p-glc-activated donors are transferred to the Man 5 GlcNAc 2 -PP-dolichol to form the core oligosaccharide Glc 3 Man 9 GlcNAc 2 -PP-dolichol (Herscovics and Orlean, 1993; Burda and Aebi, 1999; Gemmill and Trimble, 1999; Weerapana and Imperiali, 2006). Subsequently, the oligosaccharyltransferase (OST) multi-subunit complex catalyzes transfer of the core oligosaccharide to an asparagine residue of the consensus Asn-X-Ser/Thr target sequences of a nascent peptide via N-linkage. Trimming by glucosidases and mannosidase I in the ER produces various mannosetype N-glycans, which are modified in the Golgi by mannosidases and N-acetylglucosaminyltransferases (GnTI, GnTII) and other glycosyltransferases to form complex-type N-glycan structures. In yeast, deletions of some genes in the earlier steps of N-glycosylation lead to lethal or extremely severe phenotypes, indicating the biological importance of N-linked protein glycosylation. For instance, alg1, alg2 and alg11 mutants, in which synthesis of Man 5 GlcNAc 2 -PPdolichol is affected, are non-viable or grow slowly (Herscovics and Orlean, 1993; Burda and Aebi, 1999; Gemmill and Trimble, 1999; Cipollo et al., 2001; Weerapana and Imperiali, 2006). In humans, mutation of the related genes results in serious diseases that are collectively termed congenital disorders of glycosylation (Aebi and Hennet, 2001). Compared with studies in yeast, studies on the biological roles of N-linked glycosylation in plants are quite limited, partly because of the difficulties in establishing a suitable genetic or biochemical system to analyze them (Lerouge et al., 1998). Recently, a dolichol biosynthesis gene LEW1 (leaf wilting 1) was identified in Arabidopsis during a genetic screening for leaf-wilting phenotype (Zhang et al., 2008). Mutation of LEW1 reduces dolichol content, impairs protein glycosylation, and affects the plant s response to abiotic stresses. Although the LEW1 mutation does not apparently influence the cell wall, mutations in several other genes involved in N-glycan processing result in cell-wall defects, reflecting either reduced cellulose content or changes in non-cellulosic composition. For example, CYT1 (cytokinesis-defective 1) encodes a mannose-1-phosphate guanylyltransferase for synthesis of GDP-Man in the dolichol pathway. Mutation in CYT1 results in impairment of N-glycosylation and cellulose deficiency (Lukowitz et al., 2001). GCS1/KNF encodes a glucosidase I, which trims the terminal glucose of N-glycans (Boisson et al., 2001). RSW3 encodes the a-subunits of glucosidase II, which further trims the next glucosyl residue of N-glycans after GCS1/KNF (Burn et al., 2002). A mutation in either GCS1/KNF or RSW3 strongly reduces cellulose content. However, mutations in DGL1 (Defective Glycosylation 1) encoding an Arabidopsis homolog subunit of an OST complex also impair protein N-glycosylation, but the cell-wall defects of the mutants do not result from change in cellulose content (Lerouxel et al., 2005), suggesting that cell-wall defects may sometimes be related to cell-wall components other than cellulose. N-glycosylation facilitates correct protein folding, and interference with protein N-glycosylation leads to accumulation of misfolding proteins in the ER lumen, which activates an unfolded protein response (UPR) pathway. To assist folding and assembly of nascent proteins and to relieve ER stress, UPR up-regulates the expression of chaperone proteins such as the binding protein BiP. Mutation in LEW1, ALG3 (encoding an a-1,3-mannosyl transferase) or the OST complex subunit encoded by STT3a results in higher expression of the gene encoding BiP (Koiwa et al.,

3 Protein N-glycosylation, plant development and abiotic stress ; Henquet et al., 2008; Zhang et al., 2008). Interestingly, over-expression of BiP in transgenic tobacco greatly improved plant drought tolerance (Alvim et al., 2001). These results suggest that UPR and the abiotic stress pathway are closely connected (Zhang et al., 2008). In this study, we isolated a lew3 (leaf wilting 3) mutant of Arabidopsis thaliana that shows the leaf-wilting phenotype under normal growing conditions. LEW3 encodes an a-1,2- mannosyltransferase that shows high identity with yeast ALG11, and is able to partially complement the yeast alg11 mutant. The lew3 mutation reduces protein glycosylation, impairs cellulose synthesis, and results in xylem collapse. lew3 mutants are more sensitive to salt stress and transpire less water than the wild-type. BiP transcripts are induced to higher levels by abiotic stress in the mutant than in the wildtype. LEW3 must be an essential gene because a T-DNA insertion line is lethal. Our results reveal the important roles of N-glycosylation in plant development and in plant responses to abiotic stress. RESULTS Isolation of the lew3 mutant lew3 was identified during a genetic screening for mutants showing leaf-wilting phenotypes in an EMS-mutagenized Arabidopsis M 2 population as described previously (Chen et al., 2005; Zhang et al., 2008). When grown in a greenhouse, the leaves of the lew3 mutant wilted (Figure 2b). When grown at C, lew3 plants were smaller, had shorter siliques, and were less fertile than wild-type plants (Figure 2a d). However, growth of lew3 was partially rescued at higher temperatures (25 26 C), indicating that the lew3 phenotype is influenced by temperature (Figure 2e). When grown in high humidity in a sealed glass bottle, the lew3 plants no longer wilted. After the bottle was unsealed to cause a rapid drop in humidity, lew3 stems wilted within min. In contrast, the stems of wild-type seedlings remained upright. The bent stems of lew3 plants straightened (returned to an upright, normal orientation) if left overnight in the opened bottle (Figure 2f). This stem bending and straightening was also observed for plants growing in soil at the beginning of the light cycle in the morning (data not shown). These results suggest that lew3 seedlings are unable to maintain the cell turgor required to support the stem when transpiration is suddenly increased, but can adapt to this environmental change over time. The lew3 mutant exhibited a collapsed xylem with reduced cellulose content We suspected that the stem-bending and leaf-wilting phenotypes in lew3 might be caused by a water deficiency in shoots resulting from a water transport defect in the xylem (which is mainly composed of secondary cell walls) and/or a higher transpiration rate of leaves. Because xylem morphology can greatly affect the efficiency of water transport (Chen et al., 2005), we compared the xylem of roots and stems in lew3 and wild-type plants. Cross-sections of both root and stem indicated that the xylem wall was collapsed in lew3 but not in the wild-type (Figure 3a,b). We further examined the cross-sections of the stems by electron microcopy. As shown in Figure 3(c), the xylem secondary cell wall of lew3 is thinner than that of the wild-type and is unevenly distributed. Consequently, the xylem of lew3 may not be able to withstand the negative pressure produced by water transport in xylem, and may therefore collapse. A similar phenomenon was observed in a cellulose-deficient mutant leaf wilting 2 (lew2) (Chen et al., 2005). The thinner walls and the collapse of xylem cells under moderate transpiration stress indicated a defect in cell-wall composition in lew3. We used ANKOM technology to determine the acid detergent fiber (ADF) content of seedlings (Pagan et al., 1998). As shown in Figure 3(d), the ADF content (which mainly represents cellulose and lignin) was % lower in lew3 stems versus wild-type stems, and % lower in lew3 leaves versus wild-type leaves. Analysis of the neutral sugar composition of cell walls indicated that the percentages of xylose and glucose in cell walls from stems of the lew3 mutant were significantly reduced, while the percentages of arabinose and galactose were significantly increased compared to the wild-type (Figure 3e). In contrast, the percentages of mannose and rhamnose were similar in lew3 and the wild-type (Figure 3e). No apparent alterations in lignin deposition were observed in lew3 mutants when cross-sections were stained with phloroglucinol HCl, a diagnostic stain for lignin (Figure 3a). Because glucose is the major sugar in cellulose and xylose is the major sugar in hemicellulose of the cell wall, we conclude that the lew3 mutation greatly affects biosynthesis of both cellulose and hemicellulose. However, the lew3 mutation evidently does not affect biosynthesis of lignin. The increased fraction of arabinose and galactose in the neutral sugar analysis is ascribed to the reduced xylose and glucose levels in the lew3 lysates. Leaf wilting in lew3 is not due to transpiration or osmolality In order to determine whether leaf wilting is caused by high transpiration in lew3, we then measured water loss of detached leaves. The leaves were taken from 3-week-old seedlings grown in soil in a growth room under long-day conditions (16 h light/8 h dark). Unexpectedly, the water loss from detached leaves was a little slower in lew3 than in the wild-type (Figure 4a,b). We examined whether differences in the water loss rate were due to differences in stomatal aperture. Interestingly, under normal conditions (16 h light/ 8 h dark, 22 C, relative humidity 70%), the stomatal apertures of lew3 were smaller than those of the wild-type (Figure 4c,d). When detached leaves were placed under

4 986 Min Zhang et al. (c) (d) Figure 2. lew3 mutant phenotypes. (a, b) Wild-type and lew3 mutant grown in soil: 6-week-old mature plants; 3-week-old seedlings. Arrows indicate the wilting edges of lew3 leaves. (c) Leaf sizes of the 3-week-old wild-type and lew3 mutant plants. (d) Silique sizes of the 6-week-old wild-type and lew3 mutant plants. (e) Wild-type and lew3 seedlings grown at various temperatures for 5 weeks. (f) Stem bending in the lew3 mutant under low humidity. A wild-type seedling and a lew3 seedling were grown in a sealed bottle (0 min). The stem of lew3 had bent (arrow) 30 min after the bottle was unsealed, but straightened again after 12 additional hours. (e) (f) relative low humidity (30% v/v, 22 C) for 30 min, the stomatal apertures of lew3 became similar to those of the wild-type (Figure 4c,d). We also measured water loss of detached leaves taken from plants under 100% humidity conditions, and found that detached leaves of lew3 lost a similar amount of water to wild-type (Figure 4e). These results indicate that the reduced water loss of lew3 under normal conditions is due to partially pre-closed stomata, rather than a more sensitive response of stomatal movement to water loss in lew3 compared to wild-type. The smaller stomatal aperture in lew3 suggests that this mutant plant experiences water stress under normal conditions. A second means by which plants can relieve the stress of water loss is by increasing the osmolality of the cytosolic fluid. Indeed, the osmolality was much higher in lew3 than in the wild-type plants (Figure 4f). This higher osmolality was consistent with a higher content of sucrose and fructose or total soluble sugars in lew3 than in the wildtype (Figure 4g). Based on these results, leaf wilting under normal conditions or stem bending in response to a reduction in environmental humidity are the results of retarded water transport caused by (partially) collapsed xylems in lew3. The resulting water stress in lew3 is compensated for by increased osmotic potential and partial closure of stomata.

5 Protein N-glycosylation, plant development and abiotic stress 987 (d) (e) (c) Figure 3. Stem and root xylem in the wild-type and lew3 mutant. Cross-sections of stem vascular bundles in 6-week-old wild-type and lew3 plants. Lignin was stained with phloroglucinol HCl. Scale bars = 40 lm. Cross-sections of roots in 5 6-week-old wild-type and lew3 plants. Scale bars = 50 lm. (c) Stem xylem of wild-type and lew3 plants (transmission electron micrographs). Note the collapsed and uneven xylem cells in lew3. Scale bars = 5 lm. (d) ADF content (acid detergent fiber, mainly cellulose and lignin) in cell walls of wild-type and lew3 plants. (e) Neutral sugar composition [glucose, xylose, mannose (Man), rhamnose (Rham), arabinose (Arab) and galactose (Gal)] of the cell walls of the wild-type and lew3 plants. The results in (d) and (e) were obtained from three independent experiments. Data are means SE (*P < 0.05, **P < 0.01 for wild-type versus the lew3 mutant). lew3 is more sensitive to osmotic stress and ABA When seeds were sown on MS medium, lew3 showed no difference in seed germination and seedling growth compared with the wild-type. However, when seeds were sown on MS medium supplemented with 150 mm NaCl or 300 mm mannitol, the seed germination of lew3 was greatly delayed and the cotyledon greening rate of lew3 was decreased compared with wild-type (Figure 5a,b). When 4-day-old seedlings grown on MS medium were transferred to MS medium supplemented with 150 mm NaCl for 2 weeks, the lew3 mutant plants showed more root tip swelling compared to wild-type (Figure 5c,d). However, this swollen-root phenotype was not observed for seedlings grown on MS medium supplemented with 300 mm or more mannitol (data not shown). We also tested the ABA sensitivity of lew3 in a germination assay. The results show that seed germination in the lew3 mutant was more sensitive to inhibition of germination by exogenously applied ABA than the wild-type was (Figure 5a,b). Isolation of the LEW3 gene by map-based cloning We obtained an F 2 population by crossing the lew3 mutant (Columbia gl1 accession) with the wild-type (Landsberg accession). lew3 mutants with the leaf-wilting phenotype were selected from the F 2 population for genetic mapping. Using classical SSLP markers, the LEW3 locus was localized to the bottom of chromosome 2 (Figure 6a). Further fine mapping narrowed the position of lew3 to a 73 kb region within BAC clone T7M7. After sequencing all of the ORFs from the lew3 mutant in this region and comparing the sequences obtained with those in Genbank, we found a G fi A point mutation that changed amino acid Gly148 in the second exon of AT2G40190 to Glu148 (Figure 6a). To confirm the mutation, we PCR-amplified the wild-type genomic DNA that covers the whole AT2G40190 gene, including 1963 bp upstream of the first putative ATG and 540 bp downstream of the putative stop codon TGA, and cloned it into a binary vector. The constructed vector was introduced into the lew3 mutant. We obtained 11

6 988 Min Zhang et al. (c) (d) (e) (f) (g) Figure 4. Water loss from detached leaves of the wild-type and lew3. (a, b) Water loss for detached leaves of plants grown in soil under normal conditions at various time points. Detached leaves were exposed to 30% relative humidity at 22 C for the indicated periods, and fresh leaf weights were measured (three independent experiments). Water loss is expressed as a percentage of the initial fresh weight. Scale bars = 10 lm. Error bars indicate standard deviation. (c, d) Stomatal aperture sizes of the wild-type and lew3 under normal conditions and after leaves had been detached and incubated in air for 30 min (30% relative humidity, 22 C). (e) Water loss for detached leaves of 2-week-old plants grown under 100% humidity. Detached leaves were exposed to 100% relative humidity at 22 C for the indicated periods, and fresh leaf weights were measured (three independent experiments). Water loss is expressed as a percentage of the initial fresh weight. Error bars indicate standard deviation. (f) Osmolality in 3-week-old wild-type and lew3 seedlings grown in soil under normal conditions. (g) Sugar contents in wild-type and lew3 seedlings grown in soil under normal conditions. independent T 1 lines, all of which showed wild-type phenotypes (Figure 6b, middle and bottom rows). We also cloned the AT2G40190 cdna by RT-PCR, and overexpressed it under the control of the CaMV 35S promoter in lew3 and wild-type plants. Of 13 independent lines obtained, all showed wild-type phenotypes (Figure 6b, top row). However, the transgenic wild-type lines over-expressing LEW3 showed no growth phenotype (data not shown) or altered ABA and osmotic sensitivities in seed germination (Figure 5a,b). The null mutant of LEW3 interrupted by a T-DNA insertion (SALK_106951), which was inserted in position 1406 counting from the first putative ATG of the AT2G40190, was lethal (data not shown), suggesting that LEW3 is essential for plant survival. Thus, we confirmed that AT2G40190 is LEW3. LEW3 encodes a homolog of the yeast a-1,2-mannosyltransferase AGL11 that is required for protein N-glycosylation LEW3 encodes a predicted protein that shows high identity to the previously identified a-1,2-mannosyltransferase AGL11 in yeast (32% identity over the full length, 39% identity over the putative catalytic domain). The amino acid sequence was aligned to those of ALG11 from yeast and homologs from rice and humans (Figure 6d). ALG11 is a glycosyltransferase that is responsible for addition of either the 4th, the 5th, or both the 4th and 5th mannoses to form the Man 5 GlcNAc 2 -PP-Dol core oligosaccharide on the cytosolic face of the ER (Cipollo et al., 2001). Given its significant homology with ALG11, we transformed the LEW3 gene into the yeast mutant alg11, which exhibits a lowtemperature-sensitive phenotype and grows slowly at and below 30 C but normally at 37 C. As shown in Figure 6(c), expression of LEW3 partially complemented the slowgrowth phenotype of the yeast mutant alg11, suggesting that the plant ALG11 homolog is able to partially substitute for the function of ALG11 in yeast. As a positive control, we used yeast wild-type ALG11, which completely complemented the slow-growth phenotype of the yeast alg11 mutant (Figure 6c). These data clearly demonstrate that LEW3 is the ortholog of ALG11, an a-1,2-mannosyltransferase that is involved in the extension of N-glycans when they are still attached to the dolichol carrier at the cytoplasmic face of the ER. Hydrophobicity analysis predicted that LEW3 contains two transmembrane domains. In yeast, ALG11 has four putative transmembrane domains and is localized on the ER membrane (Cipollo et al., 2001). To determine the subcellu-

7 Protein N-glycosylation, plant development and abiotic stress 989 Figure 5. Phenotypic analysis of the lew3 mutant under osmotic stress and ABA treatment. (a, b) Seed germination was more sensitive to ABA, NaCl and mannitol in the lew3 mutant than in the wild-type. Seeds were germinated and grown on MS medium supplemented with 0.3 lm ABA, 150 mm NaCl or 300 mm mannitol for 2 weeks (WT, wild-type; Com, complementation plants; OE, over-expression plants). The germination greening rate of plants grown on MS medium supplemented with 0.3 lm ABA, 150 mm NaCl or 300 mm mannitol for 2 weeks. The seedlings with greening cotyledons were counted (WT, wild-type; Com, complementation plants; OE, over-expression plants). (c) The growth of lew3 seedlings was inhibited in MS medium supplemented with 150 mm NaCl. A root-swelling phenotype was observed in lew3 but not in the wild-type. (d) Root-swelling morphology of lew3 seedlings grown on MS medium supplemented with 150 mm NaCl for 7 days (light micrographs). Scale bar = 0.1 mm. (d) (c) lar localization of LEW3, we co-transfected Arabidopsis protoplasts with expression vectors containing GFP-tagged LEW3 or GFP (as a control) and RFP-tagged WAK2s (Arabidopsis thaliana wall-associated kinase 2, an established ER marker) (Nelson et al., 2007). Highly overlapping GFP and RFP fluorescence signals were observed in the protoplasts co-expressing GFP LEW3 and RFP WAK2s but not those co-expressing GFP and RFP WAK2s (Figure 7a), confirming that the LEW3 protein is localized to the ER of plants. Expression of LEW3 could not be profiled using available microarray data because the gene is not represented on microarrays from the Arabidopsis Information Resource and BAR (The Bio-Array Resource for Arabidopsis Functional Genomics). Therefore, the expression profile of LEW3 was analyzed using a LEW3 promoter::gus fusion protein. GUS staining of the transgenic lines indicated that LEW3 is expressed in the shoot, leaf, sepal, filament, silique, stem, root and guard cells (Figure 7b h). We also examined LEW3 expression using semi-quantitative and quantitative RT-PCR on RNA isolated from various organs. Figure 7(i,j) shows that LEW3 is expressed in all tissues and especially in developing stems and flowers. Effects of the lew3 mutation on N-glycosylation in Arabidopsis The effects of LEW3 on N-glycosylation were analyzed by examining the N-glycan structures on glycoproteins in the lew3 mutant. Leaves from three individual plants of lew3 and wild-type were pooled separately, and proteins were extracted. Proteins were digested with protease, and N-glycans were released from the peptides using PNGase (N-glycosidase A) A. Released N-glycans were purified and analyzed by MALDI-TOF (Table 1). There was a low level of Man 3 GlcNAc 2 and Man 4 GlcNAc 2 glycans in the lew3 sample (relative amounts of Man 3 GlcNAc 2 and Man 4 GlcNAc 2 were 0.7 and 0.8%, respectively), which were not detected in the wild-type samples. In addition, the level of Man 5 GlcNAc 2 was much reduced (14% in the wild-type versus 5% in lew3 samples), and the high-mannose-type structures Man 6-9 GlcNAc 2 were also reduced in lew3. These results are consistent with the hypothesis that LEW3 is responsible for the conversion of lipid-bound Man 3 GlcNAc 2 to Man 4 GlcNAc 2 and Man 5 GlcNAc 2. Impairment of LEW3 leads to accumulation of Man 3 GlcNAc 2 and Man 4 GlcNAc 2 lipidbound precursors, which can apparently still be flipped across the ER membrane and transferred in the ER lumen to nascent glycoproteins by the OST complex. The observation that the high-mannose-type structures Man 8 GlcNAc 2 and Man 9 GlcNAc 2 are also present on proteins demonstrates that the mutant is indeed leaky, and that a wild-type N-glycan biosynthesis pathway is also operational. Interestingly, the relative amount of complex-type glycans increased from approximately 60% in the wild-type sample to approximately 80% in the lew3 mutant. The amount of the complextype glycan Man 3 XylFucGlcNAc 2 (molecular weight 1211)

8 990 Min Zhang et al. (c) (d) Figure 6. Positional cloning of the LEW3 gene and complementation of the lew3 mutant. The LEW3 locus was mapped to the BAC clone T7M7 on chromosome 2. LEW3/At2g40190 comprises seven exons and six introns. Boxes in the LEW3 gene indicate exons and lines indicate introns. A point mutation (G fi A) in the second exon of LEW3 results in conversion of amino acid 148 from Gly to Glu. Wild-type (left), lew3 (center) and rescued lew3 (right) plants grown in soil for 3 weeks (top row) or 10 days (middle and bottom rows) after germination. Top right, lew3 transformed with 35S-LEW3 cdna; middle right, lew3 transformed with LEW3 genomic DNA. Bottom row, close-up view of single plants from the middle row. (c) LEW3 partially complemented the constitutively slow-growing phenotype of the yeast alg11 mutant. alg11 cells were transformed with an empty vector, yeast ALG11,orLEW3. Serial dilutions were spotted onto YPD (Yeast Peptone Dextrose) plates containing 10 lg ml )1 doxycycline. Plates were incubated at 25, 30 or 37 C for 3 days. WT, yeast wild-type strain R1158. (d) Alignment of LEW3 from various organisms. ABA99697, Oryza sativa (japonica cultivar group); YNL048W, Saccharomyces cerevisiae; AAI11023, Homo sapiens. Identical and similar amino acid residues are marked with letters and blot. increased from 16.3% in the wild-type to 22.9% in lew3, and that of GlcNAc 2 Man 3 XylFucGlcNAc 2 (molecular weight 1617) increased from 28% in the wild-type to 39% in lew3. GlcNAcMan 4 XylFucGlcNAc 2 (molecular weight 1576) was detected in the wild-type but remained below the level of detection in the lew3 mutant. Similar results were obtained with biological replicate samples. The (glyco)protein profiles of wild-type and lew3 mutant plants were compared using several methods (Figure 8). Western blot analysis with rabbit anti-horseradish peroxidase (HRP) antibodies, which are mostly directed against plant complex glycans (Faye et al., 1993), resulted in a slightly lower intensity of some individual complex-type glycan proteins in the lew3 mutant line compared to wildtype plants. However, qualitative differences were also

9 Protein N-glycosylation, plant development and abiotic stress 991 (e) (f) (c) (g) (h) (d) (i) (j) Figure 7. Cellular localization of LEW3 GFP and gene expression analysis of LEW3. LEW3 co-localized with an ER marker protein, WAK2. Arabidopsis protoplast cells were co-transfected with expression vectors containing GFP-tagged LEW3 or GFP alone (as a control) and RFP-tagged WAK2s. (b h) Histochemical analysis of GUS expression in LEW3 promoter:gus transgenic plants. Two-week-old seedling showing GUS expression pattern. (c h) GUS expression in various organs: (c, d) roots; (e) leaf; (f) flower; (g) guard cells; (h) shoot with flowers. Scale bars = 10 lm. (i) Semi-quantitative RT-PCR analysis of LEW3 gene expression. Total RNA was isolated from root, stems, leaves and flowers. The expression level of the rrna gene was used as a loading control. (j) Quantitative RT-PCR analysis of LEW3 expression in root, stem, leaf and flower.

10 992 Min Zhang et al. Table 1 Relative amounts of N-glycans detected in samples of pooled leaves from individual Arabidopsis thaliana wild-type or lew3 mutant plants m/z (M + Na) + Percentage of total Wild-type lew3 Hybrid and complex structures Man 3 XylGlcNAc Man 3 XylFucGlcNAc GlcNAcMan 3 XylGlcNAc GlcNAcMan 3 XylFucGlcNAc GlcNAc 2 Man 3 XylGlcNAc GlcNAcMan 4 XylFucGlcNAc ND GlcNAc 2 Man 3 XylFucGlcNAc High-mannose structures Man 3 GlcNAc ND 0.7 Man 4 GlcNAc ND 0.8 Man 5 GlcNAc Man 6 GlcNAc Man 7 GlcNAc Man 8 GlcNAc Man 9 GlcNAc Similar results were obtained with biological replicate samples. The percentages were calculated from the total peak area, divided by specific peak areas in MALDI-TOF mass spectra. m/z, mass-to-charge ratio; ND, not detectable. observed (Figure 8b). Qualitative differences were also detected in Coomassie brilliant blue-stained protein gels of wild-type and lew3 (Figure 8a). In addition, glycoproteins from the wild-type and lew3 were enriched by concanavalin A lectin, which binds branches of oligomannose chains on N-glycoproteins and therefore mainly binds ER-resident glycoproteins with high-mannose-type N-glycans. Coomassie brilliant blue staining of the SDS PAGE gel of these concanavalin A-isolated proteins also revealed qualitative differences in the profiles of wild-type and lew3 glycoproteins (Figure 8c). When these enriched protein fractions were digested using endoglycosidase H (Endo H), which cleaves high-mannose-type N-glycans, the protein profiles from wild-type and lew3 became more similar. One of the prominent protein bands present in the wild-type but not in lew3 was analyzed by MALDI-TOF MS (Autoflex II TOF/TOF). This protein was shown to be identical to TGG1 (b-thioglucoside glucohydrolase). TGG1 was also identified by differential N-glycosylation in the N-glycosylation deficiency mutant stt3a and a dolichol-deficient mutant lew1 (Koiwa et al., 2003; Zhang et al., 2008). The combined results indicate that the lew3 mutation affects the expression and N-glycan structures on multiple ER-resident (glyco)proteins. Effect of the lew3 mutation on N-glycosylation capacity (c) Figure 8. Protein N-glycosylation analysis in wild-type and lew3 mutant plants. SDS PAGE of total proteins from leaves of wild-type and lew3 plants using Coomassie brilliant staining. Arrows indicate the bands that differed between the wild-type and lew3. Western blot analysis of using polyclonal anti-hrp antibodies that specifically recognize complex N-glycans. (c) Concanavalin A Sepharose binding assay for glycoproteins. Total proteins extracted from the wild-type and lew3 were eluted through concanavalin A Sepharose, and the bound proteins were recovered. Aliquots (15 lg) of eluted proteins were separated on a 12% SDS PAGE gel. The arrow indicates the protein band found in the wild-type but not in lew3. This band was recovered and analyzed by MALDI-TOF MS. The amino acid sequence corresponds to that of b-thioglucoside glucohydrolase (TGG1). We investigated whether the lew3 mutation and the resulting truncated glycan precursor affect glycosylation efficiency. Mutants with reduced glycosylation often show increased tunicamycin (TM) sensitivity (Zhang et al., 2008). The antibiotic TM inhibits the enzyme GlcNAc phosphotransferase, which catalyzes the transfer of N-acetylglucosamine-1- phosphate from UDP-N-acetylglucosamine to dolichol phosphate in the first step of glycoprotein synthesis. TM reduces the overall N-glycosylation level, which directly affects the folding of secreted proteins, resulting in diminished plant growth (Koizumi et al., 1999). The sensitivity of lew3 seedling growth to TM was compared to that of wildtype. Four-day-old seedlings grown on MS agar medium were transferred to MS agar medium containing various concentrations of TM. After 7 days, lew3 seedlings were much paler than those of wild-type (Figure 9a), suggesting that lew3 seedlings are more sensitive to TM than wild-type seedlings. The mild sensitivity of lew3 to TM compared to wild-type indirectly suggests that the lew3 mutation reduces N-glycosylation capacity. The efficiency of N-glycosylation was specifically analyzed by determining N-glycan occupancy on the ERresident protein disulfide isomerase (PDI) by immunoblotting using an antibody directed against PDI (Henquet et al., 2008). ER glycoproteins are decorated with high mannose-

11 Protein N-glycosylation, plant development and abiotic stress 993 Figure 9. The lew3 mutant was sensitive to tunicamycin with reduced protein glycosylation capacity. Four-day-old seedlings grown on MS medium were moved to MS medium supplemented with various concentrations of tunicamycin Ô for 7 days. More cotyledons and/or true leaves became white in lew3 than in the wild-type grown on 0.5 or 1 lg ml )1 TM. Western blot analysis of protein disulfide isomerase (PDI) from the wildtype and the lew3 mutants. Isolated proteins were incubated with PNGase F (+P) or Endo H (+E) or without any enzyme ()) for 1 h. The reacted proteins were subjected to immunoblotting with anti-pdi. type glycans and are therefore sensitive to cleavage by PNGase F. In wild-type plants, PDI has two glycans (Henquet et al., 2008). As shown in Figure 9, PDI from wild-type plants that had been de-glycosylated by PNGase F (+P) treatment migrated slightly faster than untreated PDI in the gel. In contrast, untreated PDI from lew3 plants showed three bands in the gel. The middle band of the triplet most likely represents PDI with only one glycan, while the lowest one probably represents PDI without glycan, as it migrated with the same apparent molecular weight as PDI from wildtype plants treated with PNGase F (Henquet et al., 2008). To determine whether PDI in lew3 is decorated with aberrant Man 3 or Man 4 N-glycans resulting from the lew3 mutation, the protein fractions were treated with Endo H. In contrast to the wild-type high-mannose-type N-glycans on ER-resident proteins, truncated Man 3 or Man 4 N-glycans are insensitive to Endo H treatment as they lack the a-1,3-linked mannose residue on the 1,6 arm of the glycan. The results show that Endo H can remove the N-glycans from PDI in both wild-type and lew3 (Figure 9b), indicating that PDI in lew3 is not decorated with Man 3 or Man 4 N-glycans. Deficiency in N-glycosylation leads to the accumulation of misfolded proteins in the ER lumen, which activates the UPR (unfolded protein response) pathway. UPR has also been implicated in plant responses to drought stress (Zhang et al., 2008). To test whether the N-glycosylation deficiency in lew3 leads to an elevated UPR under abiotic stress, we monitored the expression of BiP, a marker gene of UPR, in the wild-type and lew3 mutant treated with ABA, NaCl, mannitol and drought (Figure 10a c). Under our conditions, BiP expression in wild-type plants was very low, and its induction was hardly detected over a short time (<4 h) under the various Figure 10. BiP was induced to higher levels in lew3 than in the wild-type by abiotic stress and ABA. Two-week-old seedlings on agar plates were treated with 100 lm ABA for 0.5, 1.0, 2.0 or 4.0 h before extraction of total RNA. rrna was used as a loading control. Two-week-old seedlings grown on agar plates were treated with 150 mm NaCl or 300 mm mannitol for 0.5, 1.0 or 4.0 h before extraction of total RNA. rrna was used as a loading control. (c) The shoots of seedlings (grown in soil for 3 weeks) were exposed to low relative humidity for approximately 1 h and then covered with transparent film. At 1 and 4 h, total RNA was extracted for Northern blot analysis. rrna was used as a loading control. BiP expression was highly induced even without drought treatment when lew3 plants were grown in soil. (d) Western blot analysis of KOR1 in the wildtype (WT) and lew3, stt3a and lew1 mutants. (c) (d)

12 994 Min Zhang et al. stress treatments. However, in the lew3 mutant, transcripts of BiP were easily detected at 1 h, and highly expressed at 4 h under ABA, NaCl, mannitol or drought treatment, indicating that the UPR pathway was more activated by abiotic stress and ABA treatment in the lew3 mutant than in the wild-type. Under normal growth conditions in soil (16 h light/8 h dark, 22 C, relative humidity 70%), BiP expression was induced to a higher level in lew3 than in the wild-type (Figure 10c), suggesting that lew3 plants are already stressed under these normal conditions. Salt sensitivity of lew3 does not correlate with under-glycosylation of KOR1 Protein N-glycosylation mutations may impair the glycosylation of proteins involved in osmotic regulation (Koiwa et al., 2003; Frank et al., 2008; Kang et al., 2008). For instance, KORRIGAN 1/RADIALLY SWOLLEN 2 (KOR1/ RSW2) encodes an endo-b-1,4-glucanase, a plasma-membrane glycoprotein that is necessary for cellulose biosynthesis (Nicol et al., 1998; Zuo et al., 2000; Lane et al., 2001). A previous study by Kang et al. (2008) indicated that the N-glycosylation of KOR1/RSW2 was altered in the mutants complex glycan 1 (cgl1) and staurosporin and temperature sensitive 3a (stt3a). Both altered N-glycosylation in cg1l and reduced N-glycosylation in stt3a are thought to disrupt the activity of KOR1, leading to root swelling under salt stress (Kang et al., 2008). This phenotype is similar to the root phenotype of lew3 under salt stress (Figure 5d). Therefore, the glycosylation levels of KOR1 were specifically tested on Western blots. However, no difference in electrophoretic mobility of the immunodetected KOR1 protein was observed in lew3 or lew1 (Zhang et al., 2008), but a difference was detected in stt3a (Figure 10d), suggesting that glycosylation of this plasma membrane protein was not altered in lew3 or lew1. Therefore, the effect of the lew3 mutation on salt sensitivity must be due to altered activities of other glycoproteins involved in osmotic regulation. DISCUSSION By genetic screening and map-based cloning, we characterized a new gene, LEW3, encoding a mannosyltransferase, which plays a key role in biosynthesis of the core glycan in the earlier steps of N-glycoprotein pathway. We confirmed the mutation by genomic complementation of the lew3 mutant and over-expression of LEW3 cdna in a lew3 mutant background. Based on the high identity between LEW3 from plants and ALG11 from yeast, the functioning of LEW3 in plants is likely to be similar to that of ALG11 in yeast. Therefore, LEW3 is predicted to add the last two terminal a-1,2-linked mannoses to Man 3 GlcNAc 2 -PP-Dol on the cytosolic face of the ER membrane using GDP-Man as a substrate (Cipollo et al., 2001; O Reilly et al., 2006). Incorrect N-glycan synthesis frequently leads to reduced or modified N-glycosylation, which has a variety of secondary effects. Some of the null mutants in the earlier biosynthetic steps occurring on the cytoplasmic face of the ER in the N-glycan synthesis pathway result in a lethal phenotype in yeast, suggesting essential roles for the earlier steps in core oligosaccharide assembly in viable cells (Burda and Aebi, 1999). In Arabidopsis, the null mutants in both LEW1 (for dilichol biosynthesis) (Zhang et al., 2008) and LEW3 caused by T-DNA insertions were lethal, indicating the essential roles of these two genes in plant. Here, the lew3 mutant should be a weak allele that dramatically affected plant development. The lew3 mutant showed various plant developmental defects, including a dwarf phenotype with narrow leaves, reduced fertility with the short siliques, and defective cell walls. Furthermore, seed germination and/or seedling growth of lew3 mutants were sensitive to osmotic stress and ABA. These results demonstrate the important biological roles of N-glycosylation in plants. The mannose content in cell walls did not differ between the wild-type and lew3, indicating that LEW3, a mannosyltransferase, was not directly involved in addition of mannose to cell walls. The lew3 mutation impaired the deposition of cellulose and hemicellulose on the secondary cell walls, resulting in thinner cell walls and collapsed xylem. It is therefore likely that other proteins involved in cell-wall biosynthesis are affected by the lew3 mutation. Interestingly, lew3 mutants were more sensitive to osmotic stress during seed germination and growth than the wild-type. In a previous study, we found that the lew2-1 mutant is more resistant to osmotic stress than the wildtype (Chen et al., 2005). The mutation of AtCesA8/IRX1, a cellulose synthase for secondary cell walls, in lew2-1 results in collapsed xylem cells and reduced cellulose content but increased glucose content in plant cells. Similarly, the lew3 mutant reduced cellulose content and increased the content of soluble sugars. As noted, however, the two mutants differ in their response to osmotic stress. Given that LEW3 probably modifies the N-glycosylation of proteins in both primary and secondary cell-wall biosynthesis, while LEW2 only affects the cellulose biosynthesis of secondary cell walls, the lew3 mutation might have a greater influence than the lew2-1 mutation on the plant response to abiotic stress. lew3 mutants are also more sensitive to ABA during seed germination than the wild-type. The lew3 mutation leads to impairment of cell-wall synthesis with a result of accumulating more sugars. Previous studies have suggested that sugars themselves are molecular signals that show cross-talk with ABA (Fedoroff, 2002). Several ABA biosynthesis mutants (aba1, aba2 and aba3) as well as the ABA signaling mutants abi4 and abi5 are insensitive to sugar (Arenas-Huertero et al., 2000; Huijser et al., 2000; Laby et al., 2000; Rook et al., 2001). High sugar content induces the expression of short-chain dehydrogenase/reductase

13 Protein N-glycosylation, plant development and abiotic stress 995 (SDR1) in the ABA biosynthesis pathway, which increases the ABA level (Cheng et al., 2002; Chen et al., 2005). Thus the ABA-sensitive phenotype during seed germination observed for the lew3 mutant can be partially explained by high accumulation of sugars. Some ABA signaling proteins such as ABI8 could also be modified by N- glycosylation (Brocard-Gifford et al., 2004). As the lew3 mutation reduces the protein N-glycosylation level, it could also influence the protein activities involved in the ABA signaling or biosynthesis pathways. As a result, lew3 mutant might exhibit some ABA response phenotypes such as seed germination sensitivity to ABA. We found that, under normal conditions, lew3 shows a wilted phenotype, but detached leaves lose less water than the wild-type, probably because the stomatal pores on wilted leaves of lew3 were already smaller than those of wild-type. Indeed, under high humidity conditions, the water loss from detached leaf was similar in lew3 and the wildtype, suggesting a similar short-term stomatal response in lew3 and the wild-type. TGG is an abundant protein in guard cells, and has multiple glycosylation sites. A recent study indicated that tgg1 mutants are hyposensitive to ABA inhibition of stomatal opening, but show a wild-type response to ABA-induced stomatal closure (Zhao et al., 2008; Islam et al., 2009). One of the qualitative differences between protein profiles of the wild-type and the lew3 mutant was related to TGG1, which has either altered glycosylation or stability in lew3 (Figure 8c). A reduced TGG1 level (or activity) in lew3 results in reduced stomatal aperture under normal conditions, which is not consistent with the tgg1 mutant phenotype, as lew3 appears to exhibit hypersensitivity rather than hyposensitivity to endogenous levels of ABA. However, because the lew3 mutation also leads to other complex changes in cells, including the accumulation of more sugars, these changes might compromise ABA phenotypes such as ABA inhibition of stomatal opening by reduced activity of TGG1. Our work demonstrates that the lew3 mutant is deficient in N-glycosylation; both the wild-type (Figure 1a) and a putative mutant pathway (Figure 1b) (Cipollo et al., 2001) are operational. Man 9 structures can only arise from the wild-type pathway, and the presence of Man 3 and Man 4 structures on glycoproteins (Table 1) shows that extension of the 1,3 arm of the glycan with a-1,2 linked mannose residues is impaired. Despite the aberrant truncated glycan structures that result from the lew3 mutation, these lipidlinked glycans are flipped across the membrane and are subsequently recognized and transferred to proteins by the OST complex. However, the efficiency of this transfer is reduced, as evidenced by the significant under-glycosylation of PDI. This indicates that substantial amounts of dolichol-linked truncated glycans compete with normal dolichol-linked Glc 3 Man 9 glycans as substrate for the OST complex. The relative amounts of truncated versus normal Glc 3 Man 9 glycans actually transferred to protein in the lew3 mutant cannot be assessed from our experiments. The observation that the ER resident protein PDI is almost exclusively decorated with glycans that are Endo H-sensitive (Figure 9b) shows that all of these N-glycans are extended on the 1,6 arm with (at least) the a-1,3-linked mannose (by the ALG3 enzyme), which is a requirement for EndoH sensitivity. This could be explained by assuming that these glycans are derived from the wild-type pathway (Figure 1a). However, it is also possible that these Endo H-sensitive glycans are (at least in part) derived from the mutant pathway, provided that glycans with truncated 1,3 arms (due to the lew3 mutation) can be extended on the 1,6 arm with an a-1,3-linked mannose residue by ALG3 to render them sensitive to Endo H. Indeed, it has been shown for yeast that glycans with a truncated 1,3 arm, due to an Alg11 mutation, can be extended on the 1,6 arm by the respective mannosyltransferases (Cipollo et al., 2001). Transfer of aberrant N-glycans to ER-resident proteins may affect their stability, but the glycans derived from the mutant pathway in lew3 plants can be converted to complex-type glycans in the Golgi (Figure 1b). Combined, these effects may explain the increased proportion of complex-type glycans in the mutant (Table 1). Protein under-glycosylation often leads to disruption of the ER protein homeostasis and causes ER stress, which triggers the unfolded protein response (UPR). The UPR activates expression of chaperone proteins such as BiP to assist protein folding to alleviate the ER stress (Schroder, 2008). Previous studies have indicated that UPR-related genes are important for plant responses to both biotic and abiotic stresses (Alvim et al., 2001; Wang et al., 2005). The expression of BiP has been shown to be induced by osmotic stress (Alvim et al., 2001; Koiwa et al., 2003; Zhang et al., 2008). Increasing BiP expression in transgenic tobacco or soybean enhances tolerance to both ER and drought stress (Alvim et al., 2001; Valente et al., 2009). We detected higher expression levels of BiP in lew3 mutants than in the wild-type under abiotic stress, including drought as well as osmotic stress and ABA treatment. Similarly, higher BiP expression was also observed in the lew1 and stt3a mutants than in the wildtype under osmotic stress conditions (Koiwa et al., 2003; Zhang et al., 2008). These results suggest that pathways controlling the UPR might be important targets for osmotic stress in plants (Zhang et al., 2008). However, the responses of the lew1, lew3 and stt3a mutants to osmotic stress also show differences. lew1 mutants are more tolerant of osmotic stress, while lew3 and sst3a mutants are more sensitive to osmotic stress than wildtype (Koiwa et al., 2003; Zhang et al., 2008). It appears that defects in different steps of the N-glycosylation pathway have different effects on secreted protein modifications,