Reconstitution of DPMS Activity and TLC Analysis of Dol₁₄-P-Man Produced in Vitro.
Assays were performed using purified recombinant DPMS1, DPMS2, and DPMS3 either alone (lanes 1 to 3) or in different combinations: DPMS1 + DPMS2 + DPMS3 (lane 4), DPMS1 + DPMS2 (lane 5), DPMS1 + DPMS3 (lane 6), and DPMS2 + DPMS3 (lane 7). The TLC plate was developed using the solvent mixture (dichloromethane/methanol/water; 10:10:3, v/v/v), and reaction products were analyzed by autoradiography. The origin (Or) and the position of Dol₁₄-P-Man (arrow) are indicated.

Interaction between DPMS Protein Components.
(A) N. benthamiana leaves were infiltrated with 1:1:1 or 1:1 mixtures of Agrobacterium strain GV3101 individually harboring the GFP-DPMS1, GFP-DPMS2, or GFP-DPMS3 constructs. Leaves were infected similarly with Agrobacterium harboring an empty GFP vector control. Infiltrated leaves were collected 72 h later for immunoblot analysis. Coomassie blue–stained protein bands of control (lane 1), GFP-DPMS1 + GFP-DPMS2 + GFP-DPMS3 transfected leaves (lane 2), GFP-DPMS1 + GFP-DPMS2 transfected leaves (lane 3), GFP-DPMS1 + GFP-DPMS3 transfected leaves (lane 4), and GFP-DPMS2 + GFP-DPMS3 transfected leaves (lane 5) were used for immunoprecipitation with anti-DPMS1. The anti-DPMS1 immunoprecipitates were used for immunoblot analysis with anti-GFP as shown: lane 6, immunoprecipitates from control leaves; lanes 7 to 10, immunoadsorbed fractions from leaves expressing GFP-DPMS1+ GFP-DPMS2 + GFP-DPMS3 (lane 7), GFP-DPMS1 + GFP-DPMS2 (lane 8), GFP-DPMS1 + GFP-DPMS3 (lane 9), and GFP-DPMS2 + GFP-DPMS3 (lane 10).
(B) Recombinant DPMS3 was biotinylated and used as a bait to pull down recombinant DPMS1 and DPMS2. For a negative control, Rubisco protein was used as bait. The soluble extracts obtained from E. coli overexpressing recombinant DPMS1 and DPMS2 as thioredoxin fusion proteins were incubated with purified DPMS3 or Rubisco immobilized on agarose beads for 2 h at 6°C before extensive washing and elution with acidic buffer, pH 2.8). SDS-PAGE and Coomassie blue–stained proteins from total proteins from induced bacteria harboring an empty vector (lane 1; pBAD/TOPO ThioFusion), 1:1 mixture of total proteins from induced bacteria expressing DPMS1 and DPMS2 (lane 2), DPMS3-interacting proteins (lane 3; P1, 43.8 kD and P2, 25.2 kD) that were subsequently subjected to nanoLC-MS/MS analysis; total proteins from induced bacteria expressing DPMS1 (lane 4), DPMS3-interacting protein (lane 5; P1, 43.8 kD), total proteins from induced bacteria expressing DPMS2 (lane 6), DPMS3-interacting protein (lane 7; P2, 25.2 kD), and Rubisco interacting proteins (lane 8).

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 ± 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 ± sd from triplicates. Bars = 1 cm.

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 μm for stem sections in (E) to (M), and 100 μm for seeds in (N) to (P).

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.

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-].

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 μm.

Immunoblot Analysis of Total Leaf Proteins Using Antibodies Directed against N-Linked Glycan PDI or GPI-Anchored PDCB1.
(A) Coomassie blue–stained proteins extracted from the wild type (lane 1), dpms1-1 (lane 2), and dpms1-2 (lane 3).
(B) Immunodetection of PDI in the wild type (lane 1), dpms1-1 (lane 2), and dpms1-2 (lane 3). Arrows indicate electrophoretic mobilities of polypeptides.
(C) Coomassie blue–stained proteins extracted from the wild type (lane 1), dpms1-1 (lane 2), and dpms1-2 (lane 3).
(D) Immunodetection of PDCB1 in the wild type (lane 1), dpms1-1 (lane 2), and dpms1-2 (lane 3). Arrows indicate electrophoretic mobilities of polypeptides.

Quantitative Analysis of the Expression of Dolichol and Glycan Biosynthetic Genes and Stress Marker Genes.
(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, and DPMS1-overexpressing (DPMS1-Ov1) plants. The expression values 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 ± sd of triplicates from three independent biological samples. Dol-P-Man and glycan pathway genes (zone I): GDP-Man pyrophosphorylase/Man-1-pyrophosphatase VTC1, Dolichol synthase DolS, DPMS1, DPMS2, DPMS3, mannosyltransferases ALG11 and ALG3, GPT. Glycan trimming genes (zone II): GCS1, α-glucosidase GCSL.
(B) Real-time PCR quantification of stress marker genes from 32-d-old soil-grown wild-type, dpms1-1, and DPMS1-overexpressing (DPMS1-Ov1) lines. The expression conditions are presented as in (A). UPR and ER stress markers (zone I): BZIP17, BZIP28, BZIP60, Bip2, Bip3, Calnexin, and Calreticulin. Oxidative stress markers (Zone II): RCD1 and RD29A.
(C) Real-time PCR quantification of UPR-induced marker genes following tunicamycin treatment using 21-d-old MS-grown wild-type, dpms1-1, and DPMS1-overexpressing (DPMS1-Ov1) plants. The expression values are presented relative to the ratio R = gene expression in tunicamycin-treated wild type/gene expression in untreated wild type, which is set to 1 (dashed line). Results are calculated from means values ± sd of triplicates from three independent biological samples. Dol-P-Man pathway genes (zone I): DolS, DPMS1, DPMS2, and DPMS3. UPR and ER stress markers (zone II): BZIP17, BZIP28, BZIP60, Bip2, Bip3, Calnexin, and Calreticulin.