Plcell v23 5 1985

The Plant Cell, Vol. 23: 1985–2005, May 2011, www.plantcell.org ã 2011 American Society of Plant Biologists.
All rights reserved.
DOLICHOL PHOSPHATE MANNOSE SYNTHASE1 Mediates

the Biogenesis of Isoprenyl-Linked Glycans and Influences
Development, Stress Response, and Ammonium
Hypersensitivity in Arabidopsis W
Nurul Jadid,a,b,1 Alexis Samba Mialoundama,a,1 Dimitri Heintz,a Daniel Ayoub,c Mathieu Erhardt,a Jérôme Mutterer,a
Denise Meyer,a Abdelmalek Alioua,a Alain Van Dorsselaer,c Alain Rahier,a Bilal Camara,a and Florence Bouviera,2
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a Institut
de Biologie Moléculaire des Plantes du Centre National de la Recherche Scientifique, Université de Strasbourg, 67084
Strasbourg Cedex, France
b Department of Biology, Botanical and Plant Tissue Culture Laboratory, Sepuluh Nopember Institut of Technology (Its), Gedung
H Kampus Its Sukolilo, Surabaya 60111, East-Java, Indonesia

c Laboratoire de Spectrométrie de Masse Bio-Organique, Département des Sciences Analytiques, Institut Pluridisciplinaire
Hubert Curien du Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7178, Université de Strasbourg,
67087 Strasbourg Cedex, France
The most abundant posttranslational modification in nature is the attachment of preassembled high-mannose-type glycans,
which determines the fate and localization of the modified protein and modulates the biological functions of glycosyl-
phosphatidylinositol-anchored and N-glycosylated proteins. In eukaryotes, all mannose residues attached to glycoproteins
from the luminal side of the endoplasmic reticulum (ER) derive from the polyprenyl monosaccharide carrier, dolichol
P-mannose (Dol-P-Man), which is flipped across the ER membrane to the lumen. We show that in plants, Dol-P-Man is
synthesized when Dol-P-Man synthase1 (DPMS1), the catalytic core, interacts with two binding proteins, DPMS2 and
DPMS3, that may serve as membrane anchors for DPMS1 or provide catalytic assistance. This configuration is reminiscent
of that observed in mammals but is distinct from the single DPMS protein catalyzing Dol-P-Man biosynthesis in bakers’
yeast and protozoan parasites. Overexpression of DPMS1 in Arabidopsis thaliana results in disorganized stem morphology
and vascular bundle arrangements, wrinkled seed coat, and constitutive ER stress response. Loss-of-function mutations
and RNA interference–mediated reduction of DPMS1 expression in Arabidopsis also caused a wrinkled seed coat phenotype
and most remarkably enhanced hypersensitivity to ammonium that was manifested by extensive chlorosis and a strong
reduction of root growth. Collectively, these data reveal a previously unsuspected role of the prenyl-linked carrier pathway
for plant development and physiology that may help integrate several aspects of candidate susceptibility genes to ammo-
nium stress.
INTRODUCTION N-glycosylation, C- and O-mannosylation, and the glycosyl phos-

phatidylinositol (GPI) anchor attachment (Figure 1). Although
The biological functions of more than 50% of proteins are N-glycosylations occur in different taxa, including Eukarya,
determined by the covalent attachment of carbohydrates re- Bacteria, and Archaea (Weerapana and Imperiali, 2006; Jones
ferred to as glycans, which constitute the most diverse and et al., 2009), plants apparently do not perform protein
abundant posttranslational modifications found in nature C-mannosylation and protein O-mannosylation (Furmanek and
(Apweiler et al., 1999). Data from human studies reveal that most Hofsteenge, 2000; Lommel and Strahl, 2009).
virus and pathogenic microbes bind to cells via glycoproteins In eukaryotic organisms, N-glycosylation is achieved by co- or
(Hart and Copeland, 2010). Furthermore, several clinical markers posttranslational attachment of the preassembled core glycan,
and therapeutic targets of cancer cells are glycoproteins (Zhao Glc3Man9GlcNAc2-Asn (Glc, Man, and GlcNAc refer to Glc, Man,
et al., 2006; Hart and Copeland, 2010). Four different types and N-acetylglucosamine) to the g-amido group of a specific Asn
of protein glycosylation have been characterized. These include in the glycosylation consensus sequence Asn-X-Ser/Thr. The
reaction is catalyzed by an oligosaccharyltransferase multisubunit
1 These authors contributed equally to this work. complex in the endoplasmic reticulum (ER) membranes (Yan et al.,
2 Address correspondence to florence.bouvier@ibmp-cnrs.unistra.fr. 2005). This modification favors the folding of N-glycoproteins due
The author responsible for distribution of materials integral to the to increased solubility (Molinari, 2007; Hanson et al., 2009) and
findings presented in this article in accordance with the policy described represents a key signal used by the ER-associated protein deg-
in the Instructions for Authors (www.plantcell.org) is: Florence Bouvier
(florence.bouvier@ibmp-cnrs.unistra.fr).
radation machinery to determine the fate of misfolded proteins
W
Online version contains Web-only data. (Helenius and Aebi, 2001). Indeed, just after the transfer, the glycan
www.plantcell.org/cgi/doi/10.1105/tpc.111.083634 is subject to trimming processes by glucosidases I and II to allow
1986 The Plant Cell

Figure 1. Dol-P-Man Biogenesis in Plants and Its Implication in Posttranslational Modifications.
Dolichol formed from plastid- and mevalonate-derived IPP is used for the synthesis of Dol-P-Man. The reaction is catalyzed by the DPMS complex
(DPMS1, DPMS2, and DPMS3). Dolichol is also used for the synthesis of the glycan intermediate (Man5GlcNAc2-PP-Dol). Dol-P-Man and
Man5GlcNAc2-PP-Dol are subsequently translocated to the ER lumen by unknown flippases. Dol-P-Man is subsequently used for the biosynthesis
of GPI-anchored proteins and the preassembled glycan Glc3Man9GlcNAc2, which is further transferred to the amido group of Asn in the N-glycosylation
site, Asn-X-Ser/Thr. Following the trimming process, the decision is made to retain, export, or degrade the polypeptide. Man residues (in red) are
specifically donated by DPMS for GPI anchor biosynthesis or incorporated into the glycan Glc3Man9GlcNAc2-PP- by ALG enzymes: ALG3 (Man 6th),
ALG9 (Man 7th and 9th), and ALG12 (Man 8th) are indicated. Asterisks refer to subpathways not yet elucidated in plants.
interactions between the resulting high-Man glycan and ER chap- multisubunit transamidase complex (Orlean and Menon, 2007;
erones for quality control (Figure 1). Following removal of the Ferguson et al., 2009) following their translocation across the ER
terminal Man by ER a1,2-mannosidases, the N-glycoprotein is membrane. GPI anchors permit more efficient membrane anchor-
exported by the vesicular transport system to the Golgi complex ing of proteins compared with acylated or prenylated proteins
and post-Golgi compartments where the glycan core is further because they deeply penetrate into the half hydrophobic mem-
modified (Helenius and Aebi, 2001; Lehrman, 2001; Lehle et al., brane leaflet (Orlean and Menon, 2007). GPI-anchored proteins
2006; Banerjee et al., 2007; Molinari, 2007). Mutant analyses have are crucial components of plant cell surfaces and are involved in
shown that several steps of the N-glycan synthesis pathways are the control of multiple aspects of plant development, such as
vital and contribute to plant development and defense (van der cellulose deposition, wall integrity, membrane raft structure, and
Hoorn et al., 2005; Pattison and Amtmann, 2009; Saijo et al., 2009; root growth, and are implicated in plant pathogen interactions
Häweker et al., 2010). (Borner et al., 2003; Gillmor et al., 2005; Roudier et al., 2005;
Like N-glycoproteins, the preformed GPI anchor, EtNP- Debono et al., 2009). In addition, 40% of the Arabidopsis thaliana
Man3GlcN2PI (referring to ethanolamine phosphate, Man, gluco- proteins predicted to possess GPI anchors are potentially mod-
samine, and phosphatidyl inositol) is attached to proteins by a ified by O-linked arabinogalactans (Ellis et al., 2010), which in
Plant Prenyl-Linked Glycan Biogenesis 1987
addition to their biological functions are key constituents of plant use of comparative genomic analysis to identify putative plant
gums (Pettolino et al., 2006; Seifert and Roberts, 2007). homologs of the minimum core enzyme DPMS1 and its potential
The presence of several Man residues represents a conserved protein partners, DPMS2 and DPMS3. We performed a BLAST
feature of the four types of protein-linked glycans (Figure 1). In search against the Arabidopsis database using yeast DPMS1 and
the Golgi complex, only guanosine 59-diphosphate-Man (GDP- human DPMS1, DPMS2, and DPMS3 as query sequences. We
Man) is used as the Man donor, whereas in the ER, GDP-Man and noted a significant match (BLASTP, E value = 9e-47) to the peptide
prenyl-linked mannoses are used (Helenius and Aebi, 2001). The sequence encoded by At1g20575 that was subsequently consid-
prenyl monosaccharide dolichol P-Man (Dol-P-Man) acts as the ered to be the putative Arabidopsis DPMS1 (At-DPMS1). At-
main carrier of transferred Man residues. The synthesis of Dol-P- DPMS1 displayed identity 61% to human, S. pombe (61%), S.
Man proceeds via two main steps: formation of dolichyl phos- cerevisiae (28%), and rice (Oryza sativa; 85%) homologs (see

phate (Dol-P) and addition of Man from GDP-Man. In yeast and Supplemental Figure 1 online). Using the same procedure, we
mammalian cells, Dol-P is synthesized exclusively from the retrieved putative Arabidopsis DPMS2 (At1g74340) and DPMS3
mevalonic pathway between the cytosol and the ER compart- (At1g48140) proteins that have E values of 4e-14 and 0.001 and
ments (Schenk et al., 2001). In plants, the initial steps of Dol-P sequence identity 57 and 31% to human DPMS2 and DPMS3,
synthesis operate in the plastids from deoxy xylulose phosphate- respectively (see Supplemental Figures 2 and 3 online). Using the
derived isopentenyl diphosphate (IPP) (40 to 50% of total IPP prediction program HMMTOP (http://www.enzim.hu/hmmtop/
incorporated) and IPP derived from the cytosolic mevalonic html/submit.html), we noted the presence of transmembrane
pathway (Skorupinska-Tudek et al., 2008). Subsequently, doli- domains in DPMS2 (two transmembrane helices, 7-30 and 49-
chol P-Man synthase (DPMS) located on the cytoplasmic face of 73) and DPMS3 (two transmembrane helices, 7-27 and 36-55).
the ER catalyzes the transfer of Man from GDP-Man to Dol-P, Previous studies based on the relatedness to the bacterial spore
and the resulting Dol-P-Man is translocated to the luminal side of coat forming protein SpSA have shown that human DPMS1 and S.
the ER membrane by an unknown flippase (Sanyal and Menon, cerevisiae DPMS1 have similar tertiary structure, in spite of their
2010). Based on available data from mammalian cells, the divergence (Maeda and Kinoshita, 2008). These studies indicate
translocated Dol-P-Man donates all of the Man residues used that in human and S. cerevisiae, Tyr-12, Asp-44, Asp-97, and Arg-
on the luminal side of the ER for the synthesis of the GPI anchor 212 are key active residues (Lamani et al., 2006; Maeda and
and the four mannoses of N-glycans (the 6th to the 9th Man Kinoshita, 2008). These residues are conserved in Arabidopsis
residues) (Burda et al., 1999; Frank and Aebi, 2005) (Figure 1). DPMS1 (see Supplemental Figure 1 online).
Accumulating evidence supports the existence of two classes To analyze precisely the requirements for Arabidopsis Dol-P-
of DPMS. In Saccharomyces cerevisiae (Orlean et al., 1988), Man synthesis, we used purified recombinant DPMS1, DPMS2,
Trypanosoma brucei (Mazhari-Tabrizi et al., 1996), Leishmania and DPMS3 proteins (see Supplemental Figure 4 online). Recom-
mexicana (Ilgoutz et al., 1999), and Thermoplasma acidophilum binant DPMS1 alone did not catalyze Dol-P-Man synthesis (Figure
(Zhu and Laine, 1996), DPMS functions as a homomeric enzyme 2, lane 1). Similarly, recombinant DPMS2 and DMPS3 did not
termed DPMS1. By contrast, in mammals (Maeda et al., 2000; display DPMS activity either individually or when both proteins
Maeda and Kinoshita, 2008), Schizosaccharomyces pombe were combined (Figure 2, lanes 2, 3, and 7). Dol-P-Man was
(Colussi et al., 1997), and Trichoderma ressei (Kruszewska synthesized when DPMS1 + DPMS2 + DPMS3 were associated
et al., 2000), the synthesis of Dol-P-Man requires the assembly (Figure 2, lane 4), and the reaction was linear with respect to time
of a heteromeric complex, including a catalytic DPMS1 and for 45 min. When DPMS1 and DPMS3 alone were coincubated
noncatalytic DPMS2 and DPMS3 proteins. The identification, (i.e., in the absence of DPMS2), up to 20% of the activity could be
organization, and biological role of DPMS in plants are not yet recovered (Figure 2, lane 6), whereas only 5% of the activity was
known. In this study, we analyzed how Dol-P-Man is synthesized obtained in the presence DPMS1 + DPMS2 alone (Figure 2, lane 5).
in plants and evaluated its role using overexpressing and knock- Because Arabidopsis accumulates dolichols from Dol14 to Dol23
out lines. We provide evidence that in plants, Dol-P-Man is (Jozwiak et al., 2009), and due to the paucity of data characterizing
synthesized as in mammals by three protein components Dol-P-Man, scale-up reactions were performed using a reaction
referred to as DPMS1, DPMS2, and DPMS3. DPMS1 is the mixture containing nonlabeled Dol14 to analyze further the reaction
catalytic module and is tethered or assisted by DPMS2 and product of Arabidopsis DPMS. The DEAE-cellulose fraction eluted
DPMS3. Our study based on the analysis of the loss of function with dichloromethane/methanol (7v/3v) containing ammonia (0.28
and the overexpression of DPMS1 demonstrates the central role N) and 50 mM ammonium acetate was subjected to liquid chro-
played by the Dol-P-Man pathway during Arabidopsis growth matography–electrospray ionization–mass spectrometry (LC-ESI-
and development and highlights the unexpected hypersensitivity MS) analysis to characterize the putative Dol14-P-Man product
of dpms1 mutant plants to ammonium. with an expected molecular mass of 1215. LC-ESI-MS analysis
revealed under basic conditions (pH 8) the presence of an ion
with a mass-to-charge ratio (m/z) of 1196 that had the highest
RESULTS intensity, corresponding to the dehydrated form [M-H20- H]
(Figure 3A). A [M-H]- form with m/z 1214 was also identified, but
Characterization and Functional Reconstitution of DPMS with a lower intensity, and was nevertheless the best candidate
for the identification of the Man residue and the phosphate group
Because Dol-P-Man synthesis is catalyzed by homomeric or that gave a loss of m/z 241 corresponding to [Man-PO3] (see
heteromeric (Maeda and Kinoshita, 2008) enzymes, we made Supplemental Figure 5 online). These data agree with the fact
1988 The Plant Cell
in the homozygous mutant lines, compared with wild-type plants

(see Supplemental Figures 6A to 6C online). Further analysis
using anti-DPMS1 antibodies revealed the absence of DPMS1 in
dpms1-1 and dpms1-2 mutants (see Supplemental Figure 6A
online). The capacity of each dpms mutant to catalyze the
synthesis of Dol-P-Man was tested in vitro using crude micro-
somal membranes. The synthesis of Dol-P-Man could be dem-
onstrated from microsomal membranes from dpms2 and dpms3
but was reduced to ;20 to 30% of the wild-type level (see
Supplemental Figure 7A online). By contrast, in microsomal

membranes isolated from dpms1, the synthesis of Dol-P-Man
could not be demonstrated (see Supplemental Figure 7A online).
The DPMS deficiency of dpms1 plants was restored following the
expression of DPMS1 in a dpms1 mutant background (see
Supplemental Figure 7A online). Thus, as shown above, in the
absence of DPMS1, Dol-P-Man was not synthesized, regardless
Figure 2. Reconstitution of DPMS Activity and TLC Analysis of Dol14-P-
of the presence of functional DPMS2 and DPMS3.
Man Produced in Vitro. Our data suggest that Arabidopsis DPMS1, DPMS2, and
DPMS3 may interact or form a complex in vivo. The binding of
Assays were performed using purified recombinant DPMS1, DPMS2,
DPMS1, DPMS2, and DPMS3 to each other has been previously
and DPMS3 either alone (lanes 1 to 3) or in different combinations:
demonstrated using mammalian cells transfected with epitope-
DPMS1 + DPMS2 + DPMS3 (lane 4), DPMS1 + DPMS2 (lane 5), DPMS1 +
DPMS3 (lane 6), and DPMS2 + DPMS3 (lane 7). The TLC plate was
tagged proteins (Maeda et al., 2000). To determine whether
developed using the solvent mixture (dichloromethane/methanol/water; Arabidopsis DPMS proteins function as parts of a protein com-
10:10:3, v/v/v), and reaction products were analyzed by autoradiogra- plex, we transiently coexpressed green fluorescent protein
phy. The origin (Or) and the position of Dol14-P-Man (arrow) are indicated. (GFP)-tagged Arabidopsis DPMS1, DPMS2, and DPMS3 in Ni-
cotiana benthamiana leaves. As a control, leaves were trans-
fected with Agrobacterium tumefaciens harboring an empty GFP
that dolichol-hexose-phosphate molecules are better identified vector. Total proteins of transfected leaves were solubilized with
in their dehydrated form and by the characteristic ion at m/z 241 digitonin before immunoprecipitation using antibodies directed
(Rush et al., 1998; Sheeley and Reinhold, 1998; Garrett et al., against Arabidopsis DPMS1 (anti-DPMS1) covalently linked to
2007). It has also been shown that the fragmentation of Dol-P- protein A-Sepharose. Following immunoprecipitation, bound
hexose involves a partial or total loss of the hexose and the proteins were eluted with an acidic buffer and subjected to
phosphate moieties (Rush et al., 1998; Sheeley and Reinhold, immunoblot analysis using anti-GFP (Figure 4A). Immunoblot
1998; Garrett et al., 2007). Indeed, in our study, the fragmentation analysis of the immunoprecipitates obtained using anti-DPMS1
of the Dol14-P-Man generated two intense daughter ions of m/z indicates that no immunoprecipitate was recovered from leaves
1094 and 1052, corresponding to the loss of Man and [Dol- transfected with the empty GFP vector (Figure 4A, lane 6). On the
HPO4]-, respectively, and to partial loss of Man (120 D) [Dol-PO4- other hand, the anti-GFP antibodies revealed interactions be-
(C2H3O)], which were subsequently used for multiple reaction tween DPMS1, DPMS2, and DPMS3 (Figure 4A, lane 7). Collec-
monitoring (MRM) analysis (Figures 3A and 3B). tively, these data indicate that DPMS1, DPMS2, and DPMS3
All considered, these data demonstrate that DPMS1 is the coimmunoprecipitate and probably interact in vivo. Using the
catalytic module of DPMS, but the enzyme activity requires the same protocol, one could note interactions between DPMS1
coincubation of DPMS1 with DPMS2 and DPMS3. Thus, DPMS2 +DPMS2 (Figure 4A, lane 8), DPMS1+DPMS3 (Figure 4A, lane 9),
and DPMS3 are probably orthologs of mammal DPMS2 and and DPMS2+DPMS3 (Figure 4A, lane 10). To assess the role of
DPMS3 (Maeda and Kinoshita, 2008). These data suggest that the DPMS3 further, we analyzed its interaction with DPMS1 and
catalytic activity of Arabidopsis DPMS1 is more stringently mod- DPMS2 using an in vitro pull-down assay. We used biotin-labeled
ulated by DPMS3 than by DPMS2. These observations are in good DPMS3 and ribulose-1,5-bisphosphate carboxylase/oxygenase
agreement with previous data observed in the case of mammalian (Rubisco) as a control to pull down interacting proteins from an
cells and in contrast with those reported for S. cerevisiae, where Escherichia coli extract expressing recombinant DPMS1 and
DPMS1 alone catalyzes the synthesis of Dol-P-Man without the DPMS2 as thioredoxin fusion proteins. After extensive washing
assistance of additional proteins (Orlean et al., 1988). to remove nonspecifically bound proteins, the acidic pH buffer
To characterize further the role of DPMS1, DPMS2, and eluate was subjected to SDS-PAGE analysis (Figure 4B). DPMS3
DMSP3, we searched independent insertion mutants corre- pulled down two prominent polypeptides having molecular
sponding to each locus among publicly available Arabidopsis masses of 43.8 kD (P1) and 25.2 kD (P2) (Figure 4B, lane 3) that
T-DNA mutant databases. T-DNA insertion lines of DPMS1 were excised and used for in-gel tryptic digestion and sequence
(dpms1-1 and dpms1-2), DPMS2 (dpms2-1 and dpms2-2), and analysis by nanoliquid chromatography-MS/MS. The MS analy-
DPMS3 (dpms3-1 and dpms3-2) were obtained. The resulting sis of interacting proteins from the pull-down assay revealed that
homozygous insertion mutants were identified by PCR, RT-PCR, DPMS2 and DPMS1 are both DPMS3 binding proteins (see
and immunoblot analyses. DPMS transcripts did not accumulate Supplemental Table 1 online). Further analysis using individually

Figure 3. UPLC-MS/MS Analysis of Dol14-P-Man Produced by the Arabidopsis DPMS Complex.
The reaction product obtained from the nonradioactive DPMS assay was subjected to DEAE-cellulose column chromatography and the fraction eluted
with dichloromethane/methanol (7:3, v/v) containing ammonia (0.28 N), and 50 mM ammonium acetate was analyzed using two MRM modes with
negative electrospray ionization (ESI-).
(A) Dol14-P-Man with the transition of m/z 1196 > 1094 corresponding to [M-H20-H-] = 1196 and the characteristic fragment of m/z = 1094 representing
partial loss of Man [Dol14-P-(C2H3O)-H-].
(B) MRM transition of m/z 1196 > 1052 corresponding to [M-H20-H-] = 1196 and to another characteristic fragment of m/z = 1052, representing the loss
of Man [Dol14-P-Man-H-].
recombinant extracts containing DPMS1 (Figure 4B, lanes 4 and DPMS1 expression (DPMS1-RNAi). We isolated three DPMS1-
5) or DPMS2 (Figure 4B, lanes 6 and 7) revealed that both interact RNAi lines and confirmed the low expression of DPMS1 by RT-
with DPMS3. The specificity was ascertained by the fact that PCR in lines 2 and 3 (see Supplemental Figure 6D online).
these polypeptides were not pulled down by Rubisco (Figure 4B, Chlorophyll content and root growth were drastically reduced in
lane 8). These data are reminiscent of the behavior of mammalian DPMS1-RNAi3 plants (Figure 6B). The extent to which the DPMS
DPMS proteins in which DPMS3 could represent the binding activity was reduced in the DPMS1-RNAi lines was evaluated
module for DPMS1 and DPMS2 (Ashida et al., 2006). using microsomal membranes, revealing background activities
Finally, the subcellular localization of DPMS protein compo- in DPMS1-RNAi2 and DPMS1-RNAi3 lines compared with wild-
nents was analyzed by confocal microscopy using GFP-tagged type plants (see Supplemental Figure 7B online). Thus, the
DPMS1, DPMS2, and DPMS3 that were transiently expressed in phenotype analysis of DPMS1-RNAi plants recapitulated that
Arabidopsis leaves. The results indicate that DPMS1 as well as observed for T-DNA dpms1 plants. Unexpectedly, upon transfer
DPMS2 and DPMS3 were localized in the ER membranes like the to soil, dpms1 and DPMS1-RNAi plants progressively lost their
GFP-tagged marker At-RTNLB2 (Nziengui et al., 2007) (Figure 5). chlorotic phenotype and accumulated chlorophylls to near wild-
type levels (Figures 6C to 6G). The root growth of soil-grown
Hypersensitivity of dpms1 Plants to Ammonium dpms1 plants was increased but remained shorter compared
with that of wild-type plants. The seed coat of dpms1 seeds
During the first step in characterizing dpms mutants, we ob- displayed a wrinkled architecture (shown in Figure 8P). Func-
served that dpms2 (dpms2-1 and dpms2-2) and dpms3 (dpms3-1 tional complementation of the dpms1 mutant phenotype was
and dpms3-2) plants were phenotypically indistinguishable from observed following transformation of dpms1 plants with the
wild-type plants when grown on Murashige and Skoog (MS, DPMS1 gene (Figure 6G). Taken together, these observations
M0221) medium or soil. On the other hand, dpms1 (dpms1-1 and suggested that endogenous factors or the composition of the MS
dpms1-2) grown on MS (M0221) exhibited a chlorotic phenotype culture medium may play an important role in dpms1 mutant
and developed short roots (Figures 6A and 6B). To analyze phenotypes. After several modifications of the mineral compo-
further the specificity of this phenomenon, we generated addi- sition of the medium, we established a correlation between the
tional mutants by RNA interference (RNAi)-mediated reduction of presence of 20.6 mM NH4+ in MS (M0221) and the appearance of
1990 The Plant Cell
chlorotic leaves. We compared the effects of MS culture media

differing predominantly in having variable NO32/NH4+ ratios.
When dpms1 mutants were cultivated on MS (M 0238) medium
(modification number 4, NH4+ free) containing exclusively NO32
(39.39 mM) as mineral nitrogen, the chlorophyll content was
retained, and the visible phenotype was indistinguishable from
that observed for the wild-type plants, except that root growth
was reduced to ;30 to 50% (see Supplemental Figure 8 online).
To test further the sensitivity and the specificity of the response
of dpms1 and DPMS1-RNAi plants to NH4+, we analyzed the

effect of increasing concentrations of NH4+ in the culture me-
dium. Cultivation on medium (MS, M 0238) containing increased
concentrations of NH4+ decreased both the chlorophyll pigments
and root growth of dpms1 and DPMS1-RNAi plants, compared
with plants grown on the same medium devoid of NH4+ (Figures
6H and 6I). The severity of the ammonium-induced effects on
dpms1 plants increased and became nonreversible and lethal
with longer exposure time. These analyses revealed that the
presence of NH4+ in MS medium was responsible for the chlo-
rotic phenotype of dpms1 plants. The chlorophyll content of wild-
type plants was not changed by any of the NH4+ concentrations
used; only root growth was slightly reduced at higher NH4+
Figure 4. Interaction between DPMS Protein Components. concentrations (Figure 6I). Thus, our data demonstrate that
functional DPMS1 is vital in the adaptation of Arabidopsis to
(A) N. benthamiana leaves were infiltrated with 1:1:1 or 1:1 mixtures of
increased external NH4+ concentration. It has been previously
Agrobacterium strain GV3101 individually harboring the GFP-DPMS1,
shown that NH4+ induces a dramatic decrease of root growth in
GFP-DPMS2, or GFP-DPMS3 constructs. Leaves were infected similarly
with Agrobacterium harboring an empty GFP vector control. Infiltrated
Arabidopsis in the absence of K+ (Cao et al., 1993). As the
leaves were collected 72 h later for immunoblot analysis. Coomassie concentration of K+ (20.04 mM) in the MS medium usually
blue–stained protein bands of control (lane 1), GFP-DPMS1 + GFP- supports optimal Arabidopsis growth, we can conclude that K+
DPMS2 + GFP-DPMS3 transfected leaves (lane 2), GFP-DPMS1 + GFP- could not alleviate the toxic effect of NH4+. Therefore, our data
DPMS2 transfected leaves (lane 3), GFP-DPMS1 + GFP-DPMS3 trans- suggest that dpms1 mutants and DPMS1-RNAi plants are NH4+
fected leaves (lane 4), and GFP-DPMS2 + GFP-DPMS3 transfected hypersensitive, regardless of the presence K+. Our data unveil a
leaves (lane 5) were used for immunoprecipitation with anti-DPMS1. The previously unsuspected connection between the synthesis of
anti-DPMS1 immunoprecipitates were used for immunoblot analysis Dol-P-Man and the sensitivity of plant to ammonium. To explore
with anti-GFP as shown: lane 6, immunoprecipitates from control leaves;
further the connection between the Dol-P-Man pathway and
lanes 7 to 10, immunoadsorbed fractions from leaves expressing GFP-
NH4+ in Arabidopsis, we analyzed the behavior of Arabidopsis
DPMS1+ GFP-DPMS2 + GFP-DPMS3 (lane 7), GFP-DPMS1 + GFP-
DPMS2 (lane 8), GFP-DPMS1 + GFP-DPMS3 (lane 9), and GFP-DPMS2 +
srd5a3-like mutants. SRD5A3 encodes the reductase catalyzing
GFP-DPMS3 (lane 10). the later steps of dolichol synthesis from polyprenol in mammals
(B) Recombinant DPMS3 was biotinylated and used as a bait to pull and yeast and probably in plants (Cantagrel et al., 2010) (see
down recombinant DPMS1 and DPMS2. For a negative control, Rubisco Supplemental Figure 9A online). The Arabidopsis genome con-
protein was used as bait. The soluble extracts obtained from E. coli tains two Arabidopsis SRD5A3-LIKE genes, At2g16530 and
overexpressing recombinant DPMS1 and DPMS2 as thioredoxin fusion At1g72590 (see Supplemental Figure 10 online). We analyzed
proteins were incubated with purified DPMS3 or Rubisco immobilized on two independent homozygous knockout lines, srd5a3-like1-1
agarose beads for 2 h at 68C before extensive washing and elution with and srd5a3-like1-2, corresponding to At2g16530 (see Supple-
acidic buffer, pH 2.8). SDS-PAGE and Coomassie blue–stained proteins
mental Figures 9B and 9C online). We found that srd5a3-like1
from total proteins from induced bacteria harboring an empty vector
and srd5a-like2 display chlorotic phenotypes remarkably similar
(lane 1; pBAD/TOPO ThioFusion), 1:1 mixture of total proteins from
induced bacteria expressing DPMS1 and DPMS2 (lane 2), DPMS3-
to those observed for dpms1 (Figures 6A and 6B) when grown in
interacting proteins (lane 3; P1, 43.8 kD and P2, 25.2 kD) that were the presence of ammonium (see Supplemental Figures 9D and
subsequently subjected to nanoLC-MS/MS analysis; total proteins from 9E online). Although additional work is needed to evaluate fully
induced bacteria expressing DPMS1 (lane 4), DPMS3-interacting protein the role of Arabidopsis SRD5A3-LIKE genes, these results ex-
(lane 5; P1, 43.8 kD), total proteins from induced bacteria expressing tend the fact that knockout of DPMS1 potentiates ammonium
DPMS2 (lane 6), DPMS3-interacting protein (lane 7; P2, 25.2 kD), and sensitivity in Arabidopsis. Previous studies established a link
Rubisco interacting proteins (lane 8). between GDP-Man pyrophosphorylase (GMPase) and ammo-
nium sensitivity in Arabidopsis using vtc1 and hsn, two allelic
GMPase-deficient mutants (Qin et al., 2008; Barth et al., 2010).
Therefore, we determined the GMPase activity of wild-type
and dpms1 mutant plants. The GMPase activity of wild-type
plants (940 6 50 nmol/h/mg protein) and dpms1 plants (915 6 30

Figure 5. ER Localization of Arabidopsis DPMS Protein Components.
Arabidopsis leaves were transfected using GFP-tagged DPMS1, DPMS2, or DPMS3 (GFP-DPMS1, GFP-DPMS2, and GFP-DPMS3). The reticulon-like
GFP-AtRTNBLB2 (Nziengui et al., 2007) was used as a positive marker of the ER membranes. Chloroplast autofluorescence is shown in red. Bars = 10 mm.
nmol/h/mg protein) was nearly identical. Because ascorbic acid reactive polypeptides suggests a modified glycosylation status
(AsA) is produced via GMPase activity, we determined the AsA between wild-type plants and dpms1 mutants (see Supplemen-
content of wild-type and dpms1 plants. The AsA content of tal Figure 11 online). Decreased Dol-P-Man availability makes
dpms1 plants (2700 6 200 nmol/g fresh weight) was lower than it likely that N-glycosylation and GPI anchor attachment are
that of wild-type plants (3500 6 300nmol/g fresh weight). How- perturbed or impaired. We therefore tested both types of post-
ever, when dpms1 plants were supplemented with 5 mM translational modifications using anti-Protein Disulfide Isomer-
galactono-1,4-lactone, the proximal AsA precursor, the wild- ase (PDI) (Lukowitz et al., 2001) and anti-Plasmodesmata Callose
type phenotype was not restored. Therefore, our data indicate Binding (PDCB1) protein (Simpson et al., 2009) antibodies as
that AsA deficiency could not account for the dpms1 mutant genuine markers of N-glycosylated and GPI-anchored proteins,
phenotype. In addition, the data suggest that when Dol-P-Man is respectively. Immunoblot analysis revealed that PDI migrated
not synthesized in Arabidopsis, the resulting GDP-Man precursor slightly faster in dpms1-1 and dpms1-2 mutants than in the wild-
is not redirected toward the synthesis of AsA. Thus, downstream type plants, consistent with a modified N-glycosylation pattern
steps involving the use of Dol-P-Man are probably key deter- (Figures 7A and 7B). On the other hand, for PDCB1 we detected
minants of Arabidopsis NH4+ sensitivity. To analyze further the in the wild type several polypeptide bands with a molecular mass
consequence of DPMS1 deficiency, total proteins from wild-type of 19 to 22 kD (Figures 7C and 7D) due to the fact that PDCB is
and dpms1 were subjected to affinodetection using using con- encoded by a multigene family in Arabidopsis (Simpson et al.,
canavalin A, a high Man-specific lectin (Faye and Chrispeels, 2009). In dpms1-1 and dpms1-2, the lower molecular mass
1985). Several reactive bands corresponding to glycoproteins PDCBs was not detected, whereas the content of the higher
bearing N-linked glycans could be observed in the wild-type and molecular mass species was reduced, thus suggesting a GPI
in the dpms1 mutant. The different electrophoretic mobility of anchor deficiency (Figures 7C and 7D).
1992 The Plant Cell

Figure 6. Ammonium Sensitivity of DPMS1 Mutants.
(A) Aerial and root phenotypes of wild-type (left) and dpms1-1 (right) plants grown in vitro on MS (M0221) agar medium containing 20.6 mM NH4+ for 26 d.
(B) Phenotypes of wild-type (left column), dpms1 (middle column, from the top to bottom dpms1-1, dpms1-1, and dpms1-2), and DPMS1-RNAi (right
column, from the top to bottom, lines 2, 2, and 3) plants grown in vitro on MS (M0221) agar medium containing 20.6 mM NH4+ for 20 d.
(C) Wild-type plants first grown in vitro on MS (M0221) agar medium containing 20.6 mM NH4+ for 27 d were transferred to soil and grown for an
additional 7 d.
(D) dpms1-1 and dpms1-2 (left column, top to bottom), DPMS1-RNAi2, and DPMS1-RNAi3 (right column, top to bottom) first grown in vitro on MS
(M0221) agar medium containing 20.6 mM NH4+ for 27 d were transferred to soil and grown for an additional 7 d.
(E) and (F) dpms1-1 (E) and DPMS1-RNAi3 (F) first grown in vitro on MS (M0221) agar medium containing 20.6 mM NH4+ for 27 d were transferred to soil
and grown for an additional 14 d.
(G) Wild-type plants (Col-0), dpms1-1 mutant (dpms1-1), and dpms1-1 plants complemented with 35S:DPMS1 (Com) first grown in vitro on MS (M0221)
agar medium containing 20.6 mM NH4+ for 27 d were transferred to soil and grown for an additional 25 d.
(H) Effect of NH4+ on chlorophyll content in wild-type, dpms1-1, and DPMS1-RNAi3 plants grown for 21 d. Results represent means values 6 SD from
triplicates.
(I) Effect of NH4+ on root growth of wild-type, dpms1, and DPMS1-RNAi3 plants grown for 21 d. Results represent means values 6 SD from triplicates.
Bars = 1 cm.
Effect of Modulated Expression of DPMS1 architecture and by perturbation of the vascular bundle arrange-
ments very similar to that observed in the clavata1 mutant that is
Loss-of-function mutants and functional rescue of a mutant phe-
altered in a receptor kinase gene (Deyoung and Clark, 2008)
notype have been widely used to assess the role of plant gene
(Figures 8E to 8P). Notably, the seed coat architecture of over-
products. The limitations of this strategy using only insertional
expressing lines nearly matched that of dpms1 mutants and is
mutants have been discussed previously (Hirschi, 2003). Previous
indicative of the formation of aberrant cell walls.
data have shown that the availability of Dol-P-Man modulates the
Because Dol-P-Man is at the convergence of several pathways,
structure and the composition of S. cerevisiae cell walls (van
Berkel et al., 1999; Orłowski et al., 2007). Therefore, we decided to we examined how overexpression of DPMS1 might influence the
generate Arabidopsis lines overexpressing DPMS1 using a Ru- expression of several genes known to encode enzymes implicated
bisco promoter that possesses enhancer elements activated by in or catalyzing upstream and downstream steps of glycan syn-
several plant transcription factors. In two selected lines, the thesis. These included the biosynthetic enzyme GDP-Man pyro-
overexpression of DPMS1 occasionally resulted in altered stem phosphorylase (also known as VTC1, for vitamin C1) (Conklin
branch diameter and morphology (Figures 8A to 8D). This phe- et al., 1997), dolichol prenyltransferase (DolS) or Leaf Wilting
nomenon was paralleled by the appearance of wrinkled seed coat 1 (LEW1) (Zhang et al., 2008), UDP-N-acetylglucosamine:dolichol
DPMS and ER Stress Status
It has been shown in yeast that the alteration in the protein

glycosylation pathways alone induces a buildup of misfolded
proteins in the ER lumen and leads to ER stress. During ER
stress, the most prominent change is represented by the tran-
scriptional induction of genes controlling the pathway known as
the unfolded protein response (UPR) (Ng et al., 2000). For
instance, the UPR is constitutively triggered in the dolichol
phosphate Man-deficient chinese hamster ovary (Foulquier
et al., 2002). It has also been reported that in fibroblasts from

patients suffering from congenital disorders of glycosylation
type-I, the UPR is moderately but constitutively induced (Lecca
et al., 2005). Finally, Arabidopsis lew mutants affected in the
biogenesis of dolichols are more sensitive to tunicamycin (Zhang
et al., 2008), which is an inhibitor of N-acetylglucosamine trans-
ferase that acts as a potent inducer of the UPR (Martı́nez and
Chrispeels, 2003; Iwata and Koizumi, 2005). Thus, due to the
central role of Dol-P-Man in protein glycosylation, we tested
whether DPMS1-deficient or DPMS1-overexpressing Arabidop-
sis plants have an altered UPR in the presence or absence of
tunicamycin. We analyzed the expression of several ER stress
marker genes, including UPR marker genes using soil-grown
plants or plants treated with tunicamycin. Marker genes included
three bZIP membrane-bound transcription factors (bZIP17,
bZIP28, and bZIP60) that globally regulate the induction of ER
stress response genes (Iwata and Koizumi, 2005; Che et al.,
2010; Liu and Howell, 2010), including the UPR markers Bip2,
Bip3, calnexin, and calreticulin. Gene expression monitored by
real-time RT-PCR revealed that UPR marker genes were con-
stitutively expressed in DPMS1-overexpressing plants compared
Figure 7. Immunoblot Analysis of Total Leaf Proteins Using Antibodies
with dpms1 and wild-type plants (Figure 9B). The expression
Directed against N-Linked Glycan PDI or GPI-Anchored PDCB1. profile of DPMS1-overexpressing plants was paralleled by
the induced expression of two transcriptional regulators of
(A) Coomassie blue–stained proteins extracted from the wild type (lane
oxidative stress responses, RCD1 (Katiyar-Agarwal et al., 2006)
1), dpms1-1 (lane 2), and dpms1-2 (lane 3).
and RD29A (Liu et al., 1998), suggesting that the ER stress
(B) Immunodetection of PDI in the wild type (lane 1), dpms1-1 (lane 2),
and dpms1-2 (lane 3). Arrows indicate electrophoretic mobilities of
in DPMS1-overexpressing plants is balanced by concomitant
polypeptides. upregulation of antioxidant pathways. When plants were treated
(C) Coomassie blue–stained proteins extracted from the wild type (lane 1), with tunicamycin to induce the UPR, the magnitude of the
dpms1-1 (lane 2), and dpms1-2 (lane 3). response of the genes encoding bZIP17, bZIP28, and bZIP60
(D) Immunodetection of PDCB1 in the wild type (lane 1), dpms1-1 (lane transcription factors, which globally regulate the stress, was
2), and dpms1-2 (lane 3). Arrows indicate electrophoretic mobilities of lower in DPMS1-overexpressing plants compared with dpms1
polypeptides. (Figure 9C). These data are consistent with ER stress being
already present in plants overexpressing DPMS1 and suggest
phosphate N-acetylglucosamine-1-P transferase (GPT) (Koizumi that dpms1 and wild-type plants are more responsive to tunica-
et al., 1999), two mannosyltransferases called Asn-linked glyco- mycin treatment than are DPMS1-overexpressing plants.
sylation (ALG) genes, ALG11 (Zhang et al., 2009) and ALG3
(Henquet et al., 2008), and the N-linked glycan trimming enzymes,
a-glucosidase I (GCS1) and an uncharacterized a-glucosidase- DISCUSSION
like enzyme (GCSL) (Boisson et al., 2001). Real-time RT-PCR
using soil-grown plants revealed that VTC1, DolS, DPMS1, Multimodular Organization of Plant DPMS
DPMS2, DPMS3, ALG11, ALG3, GPT, and GCS1, which sequen-
tially mediate the synthesis of Dol-P-Man and its downstream Man residues transported by Dol-P play a key role in the
processing, were induced in the DPMS1-overexpressing lines biosynthesis of N-glycoproteins, O- and C-mannosylated pro-
compared with dpms1 mutants as shown by the expression profile teins, and GPI-anchored proteins. In addition, the removal of the
observed for DPMS1-Ov2 and dmps1-1 (Figure 9A). Except for terminal Man residue donated by Dol-P-Man is the last enzy-
ALG11 and ALG3, a similar trend was observed between the matic step before glycoproteins can exit the ER. If folding is
DPMS1-overexpressing lines and wild-type plants (Figure 9A). not complete at this step, the attached glycan dictates the
1994 The Plant Cell

Figure 8. Phenotypic Analysis of DPMS1 Overexpression in Arabidopsis.
(A) and (B) Close-up view of the wild type (A) and a DPMS1-overexpressing plant DPMS1-Ov1 (B).
(C) Cylindrical stem of wild-type (Col-0) plants and flattened stem of DPMS1-Ov1 (Ov1) and DPMS1-Ov3 (Ov3).
(D) Higher magnification of (C).
(E) to (G) Sections of the flattened stem of DPMS1-Ov1 stained with phloroglucinol (pink color) to detect lignin in the secondary cell walls of vascular
tissues.
(H) to (J) Stem sections of dpms1-1 plants stained as in (E) to (G).
(K) to (M) Stem sections of wild-type plants stained as in (E) to (G).
(N) to (P) Scanning electron micrographs of seed coat from wild-type plants (N) compared with wrinkled seed coats of DPMS1-Ov1 (O) and dpms1-1 (P)
plants.
Bars = 1.5 cm in (A) and (B), 0.5 cm in (C) and (D), 250 mm for stem sections in (E) to (M), and 100 mm for seeds in (N) to (P).
degradation of the polypeptide chain (Tokunaga et al., 2000; Like human DPMS1, we show that Arabidopsis DPMS1 has no
Lehrman, 2001; Molinari, 2007) (Figure 1). Although the synthesis activity when expressed in E. coli, thereby pointing to a functional
of Dol-P-Man has been studied in detail in S. cerevisiae and in difference with S. cerevisiae (Colussi et al., 1997; Maeda and
human cells, the protein components catalyzing its synthesis in Kinoshita, 2008). As observed in mammalian cells (Tomita et al.,
plants were unknown. Two prototypic organizations of DPMS 1998), the synthesis of Dol-P-Man could be demonstrated in vitro
have been proposed. The first is represented by the yeast using a reconstituted enzyme system containing DPMS1,
enzyme, which comprises a homomeric protein called DPMS1 DPMS2, and DPMS3 (Figure 2). To examine the contribution of
(Maeda and Kinoshita, 2008). The second type is represented by each protein, we tested the activity of DPMS1+DPMS2, DPMS1
the mammalian enzyme, which is composed of three proteins, +DPMS3, and DPMS2+DPMS3. We observed that whereas the
including DPMS1, DPMS2, and DPMS3 (Maeda and Kinoshita, combinations of DPMS1+DPMS2 and DPMS1+DPMS3 dis-
2008). We determined the component enzymes involved in the played 5 and 20% reduced synthesis of Dol-P-Man, respectively,
synthesis of Dol-P-Man from Arabidopsis. We provide evidence compared with DPMS1+DPMS2+DPMS3, the DPMS2+DPMS3
that Arabidopsis DPMS1, DPMS2, and DPMS3 are functional combination was inactive (Figure 2). Taken together, these data
orthologs of mammalian genes involved in Dol-P-Man synthesis. suggest that Arabidopsis DPMS1 represents the catalytic core of
the enzyme complex including DPMS2 and DPMS3, as shown in

mammalian cells (Maeda and Kinoshita, 2008). Consistent with
these data, we show that endogenous DPMS1, DPMS2, and
DPMS3 are compartmentalized in the ER membranes (Figure 5)
and probably form a complex in vivo as shown by pull-down
assay and affinity interaction analyses (Figure 4). Although
DPMS2 is associated with catalytically active DPMS complex,
its precise function is unknown. It has been suggested that it may
stabilize the DPMS complex (Maeda and Kinoshita, 2008). Our
data suggest that the trimerization of DPMS could be facilitated

by the fact that DPMS3 interacts with both DPMS1 and DPMS2
(Figure 4). Because DPMS2 enhances up to twofold the activity
of GPI-N-acetylglucosaminyltransferase, which is involved in the
biosynthesis of the GPI anchor, DPMS2 has also been consid-
ered as a regulator of the GPI pathway (Watanabe et al., 2000;
Maeda and Kinoshita, 2008). This led to the suggestion that the
biosynthesis of GPI and Dol-P-Man are probably coregulated
(Orlean and Menon, 2007). It has also been shown that the
binding of Dol-P is increased in the presence of DPMS2 (Maeda
et al., 1998). In mammalian cells, deficiency of DPMS1 (Sugiyama
et al., 1991; Tomita et al., 1998), DPMS2 (Maeda et al., 1998),
and DPMS3 (Ashida et al., 2006) drastically blocks or reduces
the biosynthesis of Dol-P-Man and leads to N-glycosylation de-
fects. The requirement for DPMS3 is supported by the fact that
mammalian cell DPMS3 acts as a coiled-coil protein that tethers
DPMS1 to the ER membranes (Ashida et al., 2006; Maeda and
Kinoshita, 2008; Lefeber et al., 2009). Furthermore, a single
missense mutation in mammalian DPMS3 drastically reduces the
catalytic activity of DPMS1 and leads to muscular dystrophy
(Lefeber et al., 2009). In mammalian cells, it has also been shown
that stable expression of DPMS1 requires DPMS3, whereas
DPMS2 is required for the stable expression of DPMS3 (Maeda
et al., 1998; Maeda et al., 2000; Ashida et al., 2006). Notably,
mammalian DPMS1 is degraded by the proteasome in the ab-
sence of DPMS3 (Ashida et al., 2006). Interestingly, database
(http://atted.jp/) searches revealed that in Arabidopsis, the ex-
pression of DPMS1 is correlated with that of DPMS3. DPMS3
ranks third on the list with a Pearson’s correlation coefficient of
0.56. However, in Arabidopsis, dpms2 and dpms3 mutants still
retain DMPS activity, thus pointing to a key difference between
plants and animals (see Supplemental Figure 7A online).
Figure 9. Quantitative Analysis of the Expression of Dolichol and Glycan
Biosynthetic Genes and Stress Marker Genes.
Multiple Roles for DPMS in Plants
(A) Real-time PCR quantification of dolichol and glycan biosynthetic genes
and stress marker genes from 32-d-old soil-grown wild-type, dpms1-1, The biogenesis of Dol-P-Man involves the cooperation of several
and DPMS1-overexpressing (DPMS1-Ov1) plants. The expression values plant cell compartments. Previous studies led to the isolation of
for dpms1-1 and DPMS1-Ov1 are shown relative to the wild type, which is
set to 1 (dashed line). Results are calculated from means values 6 SD of
triplicates from three independent biological samples. Dol-P-Man and
glycan pathway genes (zone I): GDP-Man pyrophosphorylase/Man-1- (C) Real-time PCR quantification of UPR-induced marker genes following
pyrophosphatase VTC1, Dolichol synthase DolS, DPMS1, DPMS2, tunicamycin treatment using 21-d-old MS-grown wild-type, dpms1-1,
DPMS3, mannosyltransferases ALG11 and ALG3, GPT. Glycan trimming and DPMS1-overexpressing (DPMS1-Ov1) plants. The expression
genes (zone II): GCS1, a-glucosidase GCSL. values are presented relative to the ratio R = gene expression in
(B) Real-time PCR quantification of stress marker genes from 32-d-old tunicamycin-treated wild type/gene expression in untreated wild type,
soil-grown wild-type, dpms1-1, and DPMS1-overexpressing (DPMS1- which is set to 1 (dashed line). Results are calculated from means
Ov1) lines. The expression conditions are presented as in (A). UPR and values 6 SD of triplicates from three independent biological samples.
ER stress markers (zone I): BZIP17, BZIP28, BZIP60, Bip2, Bip3, Dol-P-Man pathway genes (zone I): DolS, DPMS1, DPMS2, and DPMS3.
Calnexin, and Calreticulin. Oxidative stress markers (Zone II): RCD1 UPR and ER stress markers (zone II): BZIP17, BZIP28, BZIP60, Bip2,
and RD29A. Bip3, Calnexin, and Calreticulin.
1996 The Plant Cell
Arabidopsis LEW1, which encodes a cis-prenyltransferase in- been made using purified enzymes. For instance, GPI-manno-
volved in the biosynthesis of dolichol (Zhang et al., 2008). lew1 syltransferase I, which catalyzes the first incorporation of a Man
mutants display a leaf-wilting phenotype (Zhang et al., 2008). We residue into the GPI anchor, is a multicomponent protein com-
show that among the three genes encoding the Arabidopsis plex (Orlean and Menon, 2007). Data from human, yeast, Plas-
DPMS enzyme complex, only mutations in the DPMS1 gene modium falciparuman, and T. brucei reveal that the protein
encoding the catalytic module lead to an apparent phenotype, components are species specific and are not interchangeable
characterized by extensive chlorosis, reduction of root growth, (Kim et al., 2007), thus making cross-study comparisons diffi-
and a wrinkled seed coat under normal culture conditions cult. A potentially attractive possibility is that plants could syn-
(Figures 6 and 8). Thus, our data point to nonlethal phenotypes thesize dolichol or lipid-linked phosphate Man through an
when DPMSs are knocked out. Interestingly, Arabidopsis ALG3, alternative pathway. In this view, it is worth noting that the
stringent requirement for the presence of a reduced a-isoprene

which mediates the incorporation of the 6th Man of N-glycans
from Dol-P-Man, has been identified (Henquet et al., 2008; unit in Dol-P-Man can be fulfilled by a monoterpenoid derivative,
Kajiura et al., 2010). However, alg3 mutants are undistinguish- such as citronellol phosphate (Rush et al., 1993), synthetic lipid
able from the wild-type plants when grown under normal or phosphates (Wilson et al., 1993), and various derivatives (Sprung
stress conditions, including salt stress and low and high tem- et al., 2003). Finally, the Arabidopsis genome contains several
peratures. Arabidopsis ALG12/EBS4 encodes the enzyme se- genes encoding Golgi-localized nucleotide sugar transporters
quentially catalyzing the transfer of the (8th) Man residue from (GONSTs) predicted to import GDP-Man in the Golgi (Handford
Dol-P-Man to yield the glycan intermediate Man8GlcNAc2-PP- et al., 2004). Thus, one cannot exclude the transport of GDP-Man
Dol (Hong et al., 2009). In alg12/ebs4 mutants, incomplete in the ER lumen via GONST-like transporters and its use in the
glycans are transferred to polypeptides, and the degradation of biosynthesis of N-glycoproteins and GPI-anchored proteins in
defective brassinosteroid receptors is inhibited (Hong et al., dpms1 plants.
2009). Thus, in plants, mutations affecting the synthesis and the We show that the overexpression of DPMS1 could change the
attachment of protein glycans are silent or associated with architecture of Arabidopsis stems and the vascular bundle
diverse developmental and physiological deficiencies that could arrangements and provoke the formation of a wrinkled seed
lead to lethal phenotypes (Pattison and Amtmann, 2009; Zhang coat (Figure 8), consistent with the important role played by GPI-
et al., 2009). Interestingly, short or premature glycan chains anchored proteins in cell wall formation (Gillmor et al., 2005). This
could be attached to glycoproteins in plants (Henquet et al., phenomenon is paralleled by a broad induction of the expression
2008). By contrast, DPMS1 is essential for cell viability in S. of several genes encoding the enzymes catalyzing individual
cerevisiae (Orlean, 1992), whereas knockout of mice DPMS2 steps of the N-glycan pathway (Figure 9A) and is associated with
blocks the synthesis of the GPI anchor and is embryo-lethal constitutive expression of the UPR marker genes (Figure 9B).
(Nozaki et al., 1999). Several human congenital disorders asso- These modifications probably reflect excess or aberrant glyco-
ciated with Dol-P-Man biosynthesis and metabolism have been sylations. Interestingly, the incorporation of Man in the cell walls
described (Haeuptle and Hennet, 2009). Detailed analysis re- of potato tuber was reduced to 30 to 40% of the wild-type levels
vealed that in humans, Dol-P-Man deficiency gradually affects following antisense expression of GMPase (Keller et al., 1999).
two subpathways of protein glycosylation. Mild Dol-P-Man de- Consistent with these data, the reduction of dolichol-linked
ficiency due to DPMS3 mutations affects mainly O-mannosyla- oligosaccharides to 20% of the wild type results in altered cell
tion pathways and leads to a-dystroglycanopathies. On the other walls in the S. cerevisiae mutant (cwh) (van Berkel et al., 1999).
hand, severe Dol-P-Man deficiencies perturb N-glycosylation One could also note that in S. cerevisiae overexpressing the
and GPI pathways and provoke abnormal clotting and epilepsy dolichol prenyltransferase gene RER2, the synthesis of Dol-P-
(Lefeber et al., 2009). DPMS1 deficiency caused a reduction of Man and N-protein glycosylation were increased through the
PDCB1 accumulation, consistent with an impairment of GPI stimulation of several glycan biosynthetic enzymes ALG7,
anchor biosynthesis (Figures 7C and 7D). This feature is consis- ALG13, and ALG14. This trend was paralleled by the formation
tent with previous reports showing that the impairment of GPI of an aberrant cell wall due to alteration in its composition
attachment to phospholipase C in T. brucei (Garg et al., 1997) or (Orłowski et al., 2007). Our finding may also apply to the over-
yeast Gas1p (Meyer et al., 2000) leads to lumenal ER retention, expression of S. cerevisiae DPMS1 in the fungus T. reesei. This
premature secretion, or degradation of proteins. Dol-P-Man is resulted in the formation of a floccular cell wall in the transform-
the key donor for the synthesis of the core glycan of GPI anchors ants compared with the compact wall structure of the wild type
(Orlean and Menon, 2007). Arabidopsis mutants affected in the (Kruszewska et al., 1999). This change was associated with
early steps of GPI anchor synthesis have been characterized. increased glycoprotein secretions (Kruszewska et al., 1999). In a
These include peanut, which encodes GPI-mannosyl transferase similar vein, correlations between increased DPMS activity and
I that catalyzes the transfer of the first mannosyl residues to the N-glycosylation have been documented using mammalian cells
GPI anchor (Gillmor et al., 2005), and seth1 and seth2, which (Banerjee et al., 1987; Carson et al., 1990). Interestingly, it has
encode protein components of GPI-N-acetylglucosaminyltrans- been shown that S. cerevisiae and mammal DPMSs are upregu-
ferase (Lalanne et al., 2004). In contrast with dpms1, Arabidopsis lated by phosphorylation (Banerjee et al., 2005; Baksi et al.,
peanut is lethal (Gillmor et al., 2005), and seth1 and seth2 are 2008). The Ser residue identified as the site of phosphorylation is
male sterile (Lalanne et al., 2004). GPI anchor synthesis is conserved from protozoan parasites to human, including in
complex and requires at least 18 genes (Orlean and Menon, plants, as shown in the DPMS1 sequence MRKLTS (underlined
2007). Due to the complexity of the pathway, few studies have in Supplemental Figure 1 online).
DPMS1, a Candidate Susceptibility Gene for Arabidopsis GDP-GMPase, also known as VTC1, catalyzes the
Ammonium Toxicity conversion of D-Man-1-P into GDP-Man. It has been shown
recently that mutation in the Arabidopsis GDP-GMPase gene
Although primary nitrogen assimilation in plants proceeds via causes NH4+ hypersensitivity associated with the inhibition of
its reduced form (i.e., ammonium), Arabidopsis, like the other root growth (Qin et al., 2008; Barth et al., 2010). It has been
members of the Brassicaceae, is sensitive to the presence of suggested that the strong inhibitory effect of NH4+ on GMPase
ammonium in the culture medium (Britto and Kronzucker, activity could account for NH4+ hypersensitivity (Qin et al., 2008).
2002). Interestingly, an allosteric regulation of ammonium Because, DPMS1 acts downstream of GMPase and due to the
transporter has been demonstrated that involves the phos- fact that DPMS1 and dpms1 plants have the same GMPase
phorylation of the Arabidopsis ammonium transporter AMT1 activity, our data indicate the sensitivity of GMPase to NH4+ alone
to reduce ammonium absorption (Lanquar et al., 2009). We

is not sufficient to explain the NH4+ sensitivity of Arabidopsis.
show that the Dol-P-Man pathway mediated by DPMS1, Additionally, our data from dpms1 and those obtained from vtc1
DPMS2, and DPMS3 is linked to ammonium sensitivity in plants (Barth et al., 2010) suggest that AsA is not directly
Arabidopsis. Whereas the loss-of-function mutants dpms2 implicated in the NH4+ sensitivity. Thus, the NH4+ sensitivity
and dpms3 retained a wild-type phenotype, we found that the results primarily from the perturbation of Dol-P-Man biogenesis
loss-of-function mutant dpms1 and DPMS1-RNAi exacerbate and its downstream utilization, rather than at the level of the
the hypersensitivity of Arabidopsis to ammonium and, as a branch leading to AsA. Because Dol-P-man is required for the
result, the leaves become strongly chlorotic and the root growth synthesis of N-glycoproteins, GPI-anchored proteins and arabi-
is drastically reduced. The strong depletion of DPMS1 tran- nogalactan proteins, one may reasonably expect that these
script and protein coincided with background DPMS activity in different pathways are affected. GPI-anchored proteins are
DPMS1-RNAi2 and DPMS1-RNAi3 lines (see Supplemental overrepresented in lipid rafts that are relatively enriched in
Figures 6D and 7B online). phytosterols (Kierszniowska et al., 2009) that could modulate
On the other hand, dpms2 and dpms3 have ;20 to 30% of the function of mineral transporters. With respect to the chlorotic
DPMS activity and do not display a bleached phenotype. phenotype, it is worth noting that some Arabidopsis and rice
Extrapolation from in vitro data to the in vivo situation is difficult proteins destined for the plastids are posttranslationally modified
because the integration of DPMS1 in the ER membranes is by N-glycosylation (Villarejo et al., 2005; Nanjo et al., 2006;
probably not optimal in the absence of DPMS2 or DPMS3. Radhamony and Theg, 2006). Rice a-amylase, for instance, is a
Thus, the resulting topological changes are likely amplified in glycoprotein transported from the ER-Golgi compartment to
vitro and could lower the DPMS activities measured from plastids via the secretory pathway (Kitajima et al., 2009). In
dpms2 and dpms3 microsomes. Alternatively, it could be addition, it has been shown recently that the blockage of the
possible that the reduction of DPMS activity to ;20 to 30% secretory pathway by brefeldin A induces starch accumulation in
of wild-type activity did not confer sensitivity to ammonium. The Arabidopsis plastids (Hummel et al., 2010). The above data,
chlorotic phenotype disappears when dpms1 mutant plants coupled with the fact that blocking starch catabolism triggers
were grown on soil, consistent with the usually low ammonium plastid senescence (Stettler et al., 2009), suggest that further
concentration, which has been estimated to be 10 to 1000 exploration of the link between starch hydrolysis and ammonium
times lower than that of nitrate (Marshner, 1995). The mecha- susceptibility is needed.
nism underlying the hypersensitivity to NH4+ is not well under- The biological activity of several proteins is dependent on
stood. Multiple factors have been proposed to account for posttranslational modifications, among which the covalent
ammonium toxicity in plants. The toxicity has been attributed to attachment of glycans is highly prevalent. Dol-P-Man is impli-
increased proton efflux and acidification of the external medium cated as a Man donor in the formation of N-glycoproteins, GPI-
and to the reduction of dicarboxylic acid and cation concen- anchored proteins and arabinogalactan proteins, which are
trations (Barker et al., 1966; Kirkby, 1968; Raven and Smith, collectively predicted to represent more than 50% of proteins
1976). It has also been proposed that the high demand for in Arabidopsis. Our data reveal that the synthesis of Dol-P-Man
carbohydrate skeletons, such as oxaloacetate and ketoglu- is mediated by three genes that encode DPMS1, DPMS2,
tarate, for nitrogen incorporation may lead carbon shortage and DPMS3 proteins, which assemble into a functional DPMS
(Schjoerring et al., 2002). Perturbation of hormonal homeosta- complex where DPMS1 represents the catalytic module. We
sis has also been proposed (Barker, 1999). Finally, it is well show through loss of function and overexpression studies
known that the toxicity of NH4+ is exacerbated by K+ depriva- that DPMS1, in contrast with DPMS2 or DPMS3, exerts a broad
tion (Wall, 1939) and that K+ can alleviate the toxic effect of influence on various aspects of Arabidopsis development, in-
NH4+ (Cao et al., 1993). In addition to their physicochemical cluding root growth, seed architecture, vascular bundle or-
properties (i.e., charge, size, and hydration energy) (Wang et al., ganization, activation of UPR, induction of chlorosis, and
1996; White, 1996), a reciprocal influence between K+ and NH4+ sensitivity to ammonium. Given the growing implications of
has been described during their absorption (ten Hoopen et al., glycan-linked proteins in a wide range of biological functions,
2010). Indeed, the Arabidopsis potassium transporter KAT1 has including receptor–ligand interaction, signal transduction,
affinity for NH4+ (Schachtman et al., 1992). This could contrib- pathogenesis, endocytosis, and cell wall organization, further
ute to the chlorotic phenotype and the severe reduction of root understanding of how Dol-P-Man impacts the glycome net-
growth of dpms1 plants in the basal MS medium (M0221), work and influences plant development remain important
which contains 20.04 mM K+ and 20.61 NH4+ (Figure 6). challenges for future work.
1998 The Plant Cell
METHODS DPMS2, and GFP-DPMS3 in N. benthamania was performed as described

previously using 1:1:1 or 1:1 mixtures of Agrobacterium GV3101 harboring
Plant Materials and Growth Conditions the GFP constructs (Mialoundama et al., 2009). In parallel, control leaves
were infected with Agrobacterium harboring an empty vector. The leaves
Arabidopsis thaliana plants in the Columbia background were usually grown were harvested 72 h after inoculation and processed for SDS-PAGE and
on MS basal salt medium (M0221; Duchefa Biochemie) containing Suc (3%) immunoblot analysis using anti-DPMS1 and anti-GFP antibodies (Invitro-
and agar (8 g/L) as described previously (Bouvier et al., 2006). To test the gen) (used at a 1/2500 dilution).
effect of ammonium on plant growth, the seeds were germinated on MS
medium (M 0238, modification number 4, NH4+ free; Duchefa Biochemie)
Gene Expression Analysis
supplemented with increasing NH4+ concentrations as indicated in the text.
Alternatively, the seeds were grown on damp soil (Bouvier et al., 2006). For Relative expression of the different genes was evaluated by quantitative

tunicamycin treatment, 21-d-old MS-grown (M 0238) Arabidopsis plants PCR using total RNA isolated from the different plant lines using the
were transferred to the same liquid MS medium containing 5 mg/mL of NucleoSpin RNA kit (Macherey-Nagel) according to the manufacturer’s
tunicamycin for 5 h. Leaves from 2-month-old Nicotiana benthamiana instructions. Three independent plants were used, and quantitativePCR
plants grown under greenhouse conditions were used. was performed in triplicate from each biological sample. The different
gene-specific primers were designed using Probe Finder software
Screening of T-DNA Insertion Mutants (Roche) and are listed in Supplemental Table 2 online. Two micrograms
of total RNA were reverse transcribed in a total volume of 40 mL with 2.5
The T-DNA insertion mutants of Arabidopsis (Columbia ecotypes) correspond- mM oligo(dT)20, 0.5 mM deoxynucleotide triphosphate, and 400 units of
ing to the different genes were obtained. The following lines were used: Superscript III reverse transcriptase (Invitrogen). One microliter of cDNA
GK_767A10 (dpms1-1), SALK_030487C (dpms1-2), SALK_111777 (dpms2-1), was quantified using gene-specific primers in a total volume of 10 mL
SALK_111779 (dpms2-2), SALK_037120 (dpms3-1), SALK_051037 (dpms3-2), SYBR Green I Master Mix (Roche) in a Light Cycler 480 II apparatus
SALK_006421 (srd5a3-like1-1), and SALK_ 113221 (srd5a3-like1-2). Segrega- (Roche) according to the manufacturer’s instructions. The cycle threshold
tion analysis (resistant:susceptible, 3:1) based on the antibiotic resistance, (Ct) was used to determine the relative expression level using the 22DDCt
genomic PCR, and RT-PCR analyses using appropriate primers (see Supple- method (Livak and Schmittgen, 2001). The relative expression level of
mental Table 2 online) were performed to characterize the homozygous lines as each gene was analyzed using GenEx Pro software (MultiD analyses;
described previously (Bouvier et al., 2006). Antibodies raised against recom- www.gene-quantification.de/datan.html) after normalization by reference
binant Arabidopsis DPMS1, as described previously (Bouvier et al., 2006), were with the internal references TIP41-LIKE and GAPDH.
also used for immunological analysis of dpms1mutants.
Subcellular Localization of DPMS
Histological and Microscopy Analysis
Transient Expression of GFP-tagged DPMSs in Arabidopsis leaves were
Hand-cut section of Arabidopsis stems from wild and mutant plants were performed using the above-described GFP-DPMS1, GFP-DPMS2, and
treated with phloroglucinol-HCl (0.1% phloroglucinol in 20% HCl from GFP-DPMS3 constructs as described previously (Eastmond et al., 2010).
VWR International) for 2 min to stain lignin. Sections were washed with The reticulon-like protein At-RTNLB2 fused with GFP (GFP-AtRTNBLB2)
water and examined under a light microscope. The seed surface mor- was used as an ER marker protein (Nziengui et al., 2007). Leaf tissues
phology was analyzed using scanning electron microscopy without any expressing the individual GFP constructs were analyzed by confocal
special sample preparation. Mature seeds were collected and directly microscopy as described previously (Bouvier et al., 2006).
placed into a Hitachi Tabletop microscope TM-1000 and examined by
scanning electron microscopy at an operating voltage of 15 kV. Images Recombinant DPMS Proteins and in Vitro Reconstitution of
were taken using the software supplied by manufacturer. DPMS Complex
DPMS cDNAs were subcloned into a pBAD-TOPO vector using the

Molecular Cloning and Plant Transformation
pBAD/TOPO Thio fusion vector (Invitrogen) using the primers listed in
Primers used to generate the different constructs are listed in Supplemental Supplemental Table 2 online, as described previously (Bouvier et al.,
Table 2 online. For lines overexpressing DPMS1, we cloned DPMS1 cDNA 2006). After induction with 0.001% Ara, the bacterial cells containing the
into the ImpactVector 1.1 (Plant Research International) previously digested recombinant His-tagged DPMS proteins were disrupted using a French
by NcoI and BglII. The resulting expression cassette was subcloned (AscI- press in the presence of lysis buffer A (50 mM Tris-HCl, pH 7.6, 1%
PacI) in the binary vector pBIN Plus (Plant Research International) before digitonin, 0.1% dodecylmaltoside, 50 mM KCl, and 10 mM imidazole).
transformation into Agrobacterium tumefaciens strain GV3101 and infiltra- The resulting mixture was centrifuged at 10,000g for 15 min. The super-
tion according to the floral dip procedure and kanamycin selection (Clough natant was mixed with nitrilotriacetic acid (Ni-NTA) agarose affinity
and Bent, 1998). For the complementation of dpms mutant plants, genomic chromatography (Qiagen). Ni-NTA agarose beads were rinsed three
fragments corresponding to the coding region were individually inserted times batchwise with buffer B (50 mM Tris-HCl, pH 7.6, 50 mM KCl, 0.1%
into the XbaI site of the pCAMBIA-1300 vector (http://www.cambia.org/ digitonin, and 10 mM imidazole) to remove nonspecifically bound pro-
daisy/cambia/materials/vectors). T1 plants were selected on MS containing teins. The washed beads (200 mL) were subsequently mixed with 1 mL
hygromycin. For the DPMS1-RNAi construct, we made use of a short buffer C, which contained 50 mM Tris-HCl, pH 7.6, 50 mM KCl, and 5 mg/
inverted repeat of DPMS1 cDNA (hairpin). Two 87-bp fragments amplified mL of liposomes prepared from identical amounts of phosphatidylcholine
from the DPMS1 cDNA containing NcoI and BglII at their 59 termini and and phosphatidylethanolamine using a Vibra Cell sonicator instrument
EcoRI at their 39 termini were digested by EcoRI, self-ligated, and digested (Sonics and Materials) as described previously (Maeda et al., 2000). The
with NcoI and BglII. The 194-bp fragment was then ligated to the NcoI-BglII His tag–coupled proteins were eluted from the Ni-NTA agarose beads
sites of the ImpactVector 1.1. Transient expression assays were performed with buffer C containing 150 mM imidazole. In the reconstitution assay,
using GFP-DPMS1, GFP-DPMS2, GFP-DPMS3, and At-RNLB2 inserted identical amounts of recombinant DPMS1, DPMS2, or DPMS3 in buffer C
into the NcoI site of pCATsGFP vector before insertion into the binary vec- were combined. The purified proteins were used for enzyme assay and
tor (Bouvier et al., 2006). Transient coexpression of GFP-DPMS1, GFP- SDS-PAGE analysis.
In-Gel Digestion and NanoLC-MS/MS Analysis of DMPS HPLC using a Zorbax C18 column eluted using a linear gradient of sol-
Component Proteins vent A (methanol/propan-2-ol/water, 12:8:1 v/v/v) to solvent B (hexane/
propan-2-ol, 7:3 v/v) (Skorupińska-Tudek et al., 2003).
The gel bands were predigested using a MassPREP automated system
Dol-P-Man synthase was synthesized by coupling the synthesis of Dol-P
(Waters). The gel slices were washed twice in 25 mM ammonium
to that of Dol-P-Man. First, purified Dol14 (10 mg) was incubated at 258C
hydrogen carbonate and acetonitrile. The disulfide bonds were subse-
for 10 min with (15 mg) purified recombinant polyprenyl-dolichol kinase
quently reduced in 10 mM dithiothreitol (Sigma-Aldrich) at 578C, and then
cloned from Streptococcus mutans DGK (Hartley et al., 2008) in a reaction
the Cys residues were alkylated in 55 mM iodoacetamide (Sigma-
mixture (100 mL) containing 50 mM Tris-HCl, pH 7.6, 2 mM [g-32P]ATP (10
Aldrich). Following dehydration with acetonitrile, the proteins were
mCi/mmol; Hartmann Analytic), 1 mM DTT, 5 mM MgCl2, and 0.1%
cleaved in the gel using 125 ng of modified porcine trypsin (Promega),
Tween 80. Upon completion of this step, 50 mL of liposomes containing
125 ng bovine pancreas chymotrypsin (Roche), or 130 ng of Pseudomo-
purified DPMS protein components (equivalent to 20 mg) as specified in
nas fragi AspN (Promega). The peptides were extracted sequentially with

the text were added to the reaction mixture supplemented with 2 mM
1% trifluoroactic acid in acetonitrile/water (60:40 v/v) and 100% aceto-
GDP-Man, and the reaction was allowed to proceed for 30 min. Unlabeled
nitrile before concentration using a Speed Vac system. The extracts were
Dol-P-Man was prepared under the same condition except nonradioac-
analyzed using an ultra-high-pressure LC system (NanoAcquity; Waters)
tive ATP (2 mM) was used in the incubation medium. In the case of
coupled to a high-resolution nanoelectrospray-quadrupole-time of flight
Arabidopsis microsomal membranes, the leaves were homogenized in
type mass spectrometer (Maxis; Bruker Daltonics). The samples were
the medium containing 50 mM Tris-HCl, pH 7.6, 0.25 M Suc, 1 mM EDTA,
trapped on a 20 3 0.18-mm, 5-mm Symmetry C18 precolumn (Waters),
0.5% polyvinylpyrrolidone, and 5 mM 2-mercaptoethanol. The homog-
and the peptides were separated on a nanoACQUITY UPLC BEH130 C18
enate was centrifuged at 10,000g for 15 min, and the resulting superna-
column (Waters; 75 mm 3 200 mm, 1.7 mm particle size). The solvent
tant was centrifuged at 100,000g for 1 h to pellet the microsomal
system consisted of 0.1% formic acid in water (solvent A) and 0.1%
membrane fraction. The microsomal membrane fraction was suspended
formic acid in acetonitrile (solvent B). Trapping was performed during 3
in 50 mM Tris-HCl, pH 7.6, containing 0.25 M Suc and 1 mM DTT.
min at 5 mL/min with 99% of solvent A and 1% of solvent B. Elution was
Microsomal preparations equivalent to 250 mg of protein were incubated
performed at a flow rate of 450 nL/min using 6 to 35% gradient (solvent A
at 258C for 30 min in the reaction mixture containing 50 mM Tris-HCl, pH
to B) over 9 min at 458C followed by 90% (solvent B) over 1 min before the
7.6, 2 mM [g-32P]ATP (10 mCi/mmol; Hartmann Analytic), 1 mM DTT, 5
reconditioning the column using 99% of solvent A over 6 min. Maxis
mM MgCl2, Dol14 (10 mg), 0.1% Tween 80, and 2 mM GDP-Man for 30
equipped with a nanosprayer was operated in the positive ion mode.
min. The reaction products were extracted using dichloromethane/meth-
Mass calibration of the TOF was achieved prior to analysis from m/z 50 to
anol (2:1 v/v). Following centrifugation, the dichloromethane phase was
2200 using Tunemix (Agilent Technologies). Online correction of this
adsorbed onto a mini DEAE-cellulose column prepared as described
calibration was performed using two Tunemix ions (m/z 299.2945 and
previously (Camara and Monéger, 1977). The column was sequentially
922.0098) as recalibration masses. The capillary voltage was set to 4500
eluted with dichloromethane and dichloromethane/methanol (7:3 v/v),
V and the end plate offset to 2500 V. For MS/MS experiments, the system
and the fraction containing the Dol-P-Man was eluted with dichloro-
was operated with automatic switching between MS and MS/MS modes.
methane/methanol (7:3 v/v) containing ammonia (0.28 N) and 50 mM
The MS scan was performed over an m/z range of [50;2200] at 0.2 s/scan.
ammonium acetate. The eluate was concentrated and subjected to thin
The MS/MS scan time was automatically set depending on the intensity of
layer chromatography (TLC) on silica gel plate developed with dichloro-
the selected precursor peak on the MS spectrum (from 0.1 to 1.4 s/scan
methane/methanol/water (10:10:3 v/v/v), and the position of Dol14-P-Man
over the same m/z range). The three most abundant peptides, preferably
(retention factor = 0.7) was detected by autoradiography. Radioactivity
doubly and triply charged ions, were selected on each MS spectrum for
incorporated in the products was quantified by a liquid scintillation
further isolation and collision-induced dissociation using an optimized
counter. For incubation with nonradioactive ATP, the Dol-P-Man band
collision energy depending on the charge state and the m/z of the ion. The
was scraped and eluted with dichloromethane/methanol (2:1 v/v) before
selected peptides are then excluded for 0.3 min. The complete system
ultra-high-pressure liquid chromatography coupled to tandem mass
was fully controlled by Compass Hystar (Bruker). The MS data collected
spectrometry (UPLC-MS/MS) analysis.
during the analysis were processed and converted to mascot generic files
(mgf) using Compass Data Analysis 4 software (Bruker). The mgf peak list
UPLC and MS Analysis of Dol-P-Man
files were then submitted to the Mascot search engine (Matrix Sciences;
version 2.3.01) in a local server. Searches were performed against a Characterization of Dol14-P-Man recovered from the DEAE cellulose col-
composite target decoy database generated using internally developed umn was performed using UPLC-MS/MS. All analyses were performed
tools (http://msda.u-strasbg.fr) and containing the National Center for using a Waters Quattro Premier XE equipped with an ESI source and
Biotechnology Information protein sequences of Arabidopsis and com- coupled to an Acquity UPLC system (Waters) with diode array detector. UV
mon contaminants (trypsin and human keratins) downloaded on the 23rd spectra were recorded from 200 to 500 nm. Chromatographic separation
of September 2010. Searches were performed with a tolerance of 5 ppm was achieved using an Acquity UPLC BEH C18 column (100 3 2.1 mm, 1.7
for precursor ions and 0.02 D for fragment ions, allowing a maximum of mm; Waters), coupled to an Acquity UPLC BEH C18 precolumn (2.1 3 5 mm,
one missed cleavage site for trypsin and chymotrypsin and three missed 1.7 mm; Waters). The temperature of the column oven was 408C, and the
cleavages sites for AspN. Carbamidomethylation of Cys residues, oxi- injection volume was 3 mL. Eluents were methanol/acetonitrile/water (60/
dation of Met residues, and acetylation of protein N-terminal residues 30/9.99, v/v/v), basified with 1 mM ammonium acetate and 0.01% piper-
were searched as variable modifications. The spectra that yielded iden- idine (solvent A) and ethanol 99.99%; basified with 1 mM ammonium
tifications were manually inspected. acetate and 0.03% piperidine (solvent B). The flow rate was 0.31 mL/min.
Chromatographic conditions were as follows: 100% (A) for 1 min; gradient
from 100% (A) to 100% (B) for 5 min, 100% (B) and an isocratic run of 5 min
Purification of Dolichols and Assay of DPMS Activity
at 100% of B, followed by a gradient from 100% (B) to 100% (A) for 1 min to
Ginkgo biloba seeds obtained from the botanical garden (Strasbourg, the initial conditions that was maintained for 3 min. The total run time was 15
France) were used to extract and purify dolichols with chain length of 14 to min. Nitrogen generated from pressurized air in a N2G nitrogen generator
18 isoprene residues, according to published procedures (Tateyama et al., (Mistral) was used as the drying and nebulizing gas. The nebulizer gas flow
1999). The resulting dolichol fraction was further purified by preparative was set to ;50 L/h, and the desolvation gas flow to 700 L/h. The interface
2000 The Plant Cell
temperature was set at 3008C and the source temperature at 1208C. The (used at a 1/2500 dilution) (Lukowitz et al., 2001) and anti-PDCB1 (used at
capillary voltage was set at 3 kV, and the cone voltage was optimized for a 1/2500 dilution) (Simpson et al., 2009) antibodies.
each Dol-P-Man depending on the length of the isoprenoid skeleton. Full-
scan and selected ion recording analyses were used to determine the m/z
Ascorbic Acid Analysis
of Dol-P-Man. Fragmentation was performed by collision-induced disso-
ciation with argon at 1.0 3 1024 mbar. The collision energy was optimized Arabidopsis leaves were pulverized in liquid nitrogen, and the resulting
using daughter scan monitoring and MRM. MS conditions for Dol14-P-Man powder was homogenized in 1 N HClO4. Following centrifugation and
were set after optimization as follows: polarity ES-, capillary 3 kV, cone 40 V. neutralization with K2CO3, the supernatant was saved for ascorbate
Low mass and high mass resolution were 13 for both mass analyzers, ion determination as described previously (Foyer et al., 1983).
energies 1 and 2 were 0.5 V, entrance and exit potential were 2 and 1 V, and
detector (multiplier) gain was 650 V. Collision-induced dissociation of
GMPase Activity

deprotonated parent ions was accomplished with collision energy of 10 and
15 V. Daughter scan monitoring, neutral loss, and MRM permitted the Plant leaves were pulverized in liquid nitrogen, and the resulting powder
identification of Dol14-P-Man. The prominent compound was identified as a was homogenized in the extraction buffer containing 50 mM Tris-HCl, pH
deprotonated parent ion [M-H2O-H-], and the predominant daughter frag- 7.6, and 2 mM 2-mercaptoethanol. The homogenate was centrifuged at
ment ions 1094 and 1052 corresponding to partial and total Man loss were 10,000g for 10 min, and the supernatant (100 mg protein) was used for
obtained by daughter scan and subsequently used for MRM transitions. enzyme assay in a reaction mixture (200 mL final volume) containing
The combination of chromatographic retention time, parent mass, and the 50 mM Tris-HCl, pH 7.6, 2 mM 2-mercaptoethanol, 5 mM MgCl2, 2 mM
two more intense daughter ions analysis was used to selectively monitor GTP, and 2 mM D-Man. The reaction was performed at 308C for 1 h. The
Dol14-Man-P-Man (1196 > 1094) and (1196 > 1052). Data acquisition and reaction was quenched by boiling, and GDP-Man was analyzed by HPLC
analysis were performed with the MassLynx software (ver.4.1) running as described previously (Albermann et al., 2000). Protein concentration
under Windows XP professional on a Pentium PC. was determined using the Pierce bicinchoninic protein assay kit (Pierce).
Affinity Interactions of DPMS Protein Components Chlorophyll Determination

Total leaf proteins were prepared from N. benthamiana leaves expressing Total chlorophyll content was determined in 80% acetone extracts
various combinations of GFP-DPMS1, GFP-DPMS2, and GFP-DPMS3 (Lichtenthaler and Wellburn, 1983).
constructs. Leaf tissues were ground into a fine powder in liquid nitrogen
before extraction with the buffer (50 mM Tris-HCl, pH 7.6, 2 mM PMSF, 0.
Accession Numbers
2% Triton X-100, and 3% digitonin). Anti-DPMS1-Sepharose beads were
prepared using dimethylpimelimidate (Schneider et al., 1982). Proteins Sequence data from this article can be found in the Arabidopsis Genome
derived from leaf extracts were centrifuged, and the supernatant was Initiative databases under the following accession numbers: At-DPMS1
incubated for 6 h at 48C to 100 mL of anti-DPMS1-Sepharose beads in the (Arabidopsis; At1g20575), Os-DPMS1 (Oryza sativa; NP_001051751),
presence of the incubation buffer (50 mM Tris-HCl, pH 7.6, 0.1 M KCl, 2 mM Sp-DPMS1 (Schizosaccharomyces pombe; CAB11700), Hs-DPMS1
PMSF, and 0.3% digitonin). Immunoadsorbed complexes were washed by (Homo sapiens; EAW75608), Rn-DPMS1 (Rattus norvegicus; NP_001100014),
centrifugation using the same buffer containing 0.25 M KCl before elution of Tb-DPMS1 (Trypanosoma brucei; AJ866775), Lm-DPMS1 (Leish-
bound proteins with 0.1 M Gly, pH 2.5. The eluate was neutralized to pH 7.6 mania mexicana; AJ131960), Sc-DPMS1 (Saccharomyces cerevisiae;
and subjected to SDS-PAGE and immunoblot analyses using anti-GFP AAB68116), At-DPMS2 (At1g74340), Os-DPMS2 (NP_001172795), Hs-
(Invitrogen) as described previously (Bouvier et al., 2006). DPMS2 (AAH15233), Rn-DPMS2 (NP_062125) Sp-DPMS2 (NP_595676),
The pull-down assay was performed using DPMS3 as bait to pull down At-DPMS3 (At1g48140), Os-DPMS3 (EAY87536), Hs-DPMS3
putative DPMS1 and DPMS2 preys. Streptavidin agarose beads (Thermo (BAA96291), Rn-DPMS3 (NP_001102801), Cd-DPMS3 (Candida dublin-
Scientific) (200 mL) were incubated for 30 min at 258C with biotin-labeled iensis; XP_002419846), At-SRD5A3-like1 (At2g16530), At-SRD5A3-like2
DPMS3 (250 mg) prepared using the Pierce pull-down biotinylated pro- (At1g72590), Hs-SRD5A3 (NM_024592), At-RTNLB2 (At4g11220), TIP41-
tein:protein interaction kit (Thermo Scientific). Purified Rubisco protein LIKE (At4g34270), GAPDH (At1g3440), and a-Tubulin (At1g04820).
(Sigma-Aldrich) was used as negative control bait. Next, total Escherichia
coli lysates (500 mg) obtained after French press disruption and centrif-
Supplemental Data
ugation of bacterial cells expressing recombinant DPMS1 and DPMS2
were incubated for 2 h at 68C with biotinylated DPMS3 coupled to The following materials are available in the online version of this article.
streptavidin agarose in a medium containing 50 mM Tris-HCl, pH 7.6, 3%
Supplemental Figure 1. Multiple Sequence Alignment of DPMS1
digitonin, 2 mM PMSF, 0.2% Triton X-100, and 0.1 M KCl. The beads were
with Homologous Proteins.
washed by centrifugation with 50 mM Tris-HCl, pH 7.6, buffer containing 2
mM PMSF, 0.3% digitonin, and 0.25 M KCl to remove nonspecifically Supplemental Figure 2. Multiple Sequence Alignment of DPMS2
bound proteins. DPMS3 interacting proteins were eluted with the acidic with Homologous Proteins.
pH 2.8 buffer. Supplemental Figure 3. Multiple Sequence Alignment of DPMS3
with Homologous Proteins.
Affinodetection of Glycoproteins
Supplemental Figure 4. Analysis of Recombinant DPMS Proteins
Total proteins were extracted using 25 mM Tris, pH 8.3, containing 250 Expressed in E. coli.
mM Gly and 01% SDS and subjected to SDS-PAGE analysis before Supplemental Figure 5. Neutral Loss Analysis of Dol14-P-Man.
electroblot transfer onto nitrocellulose membranes. Concanavalin A
Supplemental Figure 6. Characterization of Arabidopsis dpms Mu-
binding proteins were detected using jack bean (Canavalia ensiformis)
tants and Transgenic Lines.
Concanavalin A-peroxidase (Sigma-Aldrich) and Amersham enhanced
chemiluminescence reagent. PDI and PDCB1 were used as markers for Supplemental Figure 7. Chromatographic Analysis of Dol14-P-Man
N-glycoproteins and GPI-anchored proteins and probed using anti-PDI Synthesized by Microsomal Membranes Isolated from Wild-Type
Arabidopsis, dpms Mutants, Complemented Mutants, and DPMS1- mediated protein phosphorylation of microsomal membranes in-
RNAi Lines. creases mannosylphosphodolichol synthase activity. Proc. Natl.
Supplemental Figure 8. Ammonium Sensitivity of dpms1 Compared Acad. Sci. USA 84: 6389–6393.
with Wild-Type Plants. Banerjee, S., Vishwanath, P., Cui, J., Kelleher, D.J., Gilmore, R.,
Robbins, P.W., and Samuelson, J. (2007). The evolution of
Supplemental Figure 9. Characterization and Ammonium Sensitivity N-glycan-dependent endoplasmic reticulum quality control factors
of srd5a3-like1 Mutants. for glycoprotein folding and degradation. Proc. Natl. Acad. Sci. USA
Supplemental Figure 10. Multiple Sequence Alignment of SRD5A3 104: 11676–11681.
with Homologous Proteins. Barker, A.V. (1999). Ammonium accumulation and ethylene evolution by
tomato infected with root-knot nematode and grown under different
Supplemental Figure 11. Affinodetection Analysis of Total Leaf
regimes of plant nutrition. Commun. Soil Sci. Plant Anal. 30: 175–182.
Proteins Using Concanavalin A.

Barker, A.V., Volk, R.J., and Jackson, W.A. (1966). Root environment
Supplemental Table 1. In-Gel Digestion and NanoLC-MS/MS of acidity as a regulatory factor in ammonium assimilation by the bean
DPMS3-Interacting Proteins. plant. Plant Physiol. 41: 1193–1199.
Supplemental Table 2. Primer Sequences Used for Cloning, Geno- Barth, C., Gouzd, Z.A., Steele, H.P., and Imperio, R.M. (2010). A
typing, and Expression Studies. mutation in GDP-mannose pyrophosphorylase causes conditional
hypersensitivity to ammonium, resulting in Arabidopsis root growth
inhibition, altered ammonium metabolism, and hormone homeostasis.
ACKNOWLEDGMENTS J. Exp. Bot. 61: 379–394.
Boisson, M., Gomord, V., Audran, C., Berger, N., Dubreucq, B.,
This work was supported by the Agence Nationale de la Recherche Granier, F., Lerouge, P., Faye, L., Caboche, M., and Lepiniec, L.
Program (Contract ANR-09-BLAN-0067-02) and by a doctoral fellow- (2001). Arabidopsis glucosidase I mutants reveal a critical role of
ship from the Research Program of the Ministry of Science and Tech- N-glycan trimming in seed development. EMBO J. 20: 1010–1019.
nology of Indonesia to N.J. We thank Meredith D. Hartley and Barbara Borner, G.H., Lilley, K.S., Stevens, T.J., and Dupree, P. (2003).
Imperiali from the Massachusetts Institute of Technology for the kind gift Identification of glycosylphosphatidylinositol-anchored proteins in
of E. coli overexpressing Streptococcus mutans polyprenyl-dolichol Arabidopsis. A proteomic and genomic analysis. Plant Physiol. 132:
kinase. We thank Andrew J. Maule from the John Innes Center for anti- 568–577.
PDCB1 antibodies and Chris R. Somerville from the Carnegie Institution Bouvier, F., Linka, N., Isner, J.C., Mutterer, J., Weber, A.P., and
for PDI antibodies. We thank A. Bailly, M. Kerneis, S. Staerck, and R. Camara, B. (2006). Arabidopsis SAMT1 defines a plastid transporter
Wagner for taking care of the plants. We thank the Salk Institute regulating plastid biogenesis and plant development. Plant Cell 18:
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The Plant Cell, Vol. 23: 1985–2005, May 2011, www.plantcell.org ã 2011 American Society of Plant Biologists.

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DOLICHOL PHOSPHATE MANNOSE SYNTHASE1 Mediates

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H Kampus Its Sukolilo, Surabaya 60111, East-Java, Indonesia

INTRODUCTION N-glycosylation, C- and O-mannosylation, and the glycosyl phos-

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in the homozygous mutant lines, compared with wild-type plants

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chlorotic leaves. We compared the effects of MS culture media

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DPMS and ER Stress Status

It has been shown in yeast that the alteration in the protein

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the enzyme complex including DPMS2 and DPMS3, as shown in

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METHODS DPMS2, and GFP-DPMS3 in N. benthamania was performed as described

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DPMS cDNAs were subcloned into a pBAD-TOPO vector using the

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Affinity Interactions of DPMS Protein Components Chlorophyll Determination

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