proliferation and morphogenesis of the endoplasmic ...proliferation and morphogenesis of the...

Proliferation and Morphogenesis of the EndoplasmicReticulum Driven by the Membrane Domain of3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase inPlant Cells1[OPEN]

Sergi Ferrero2,3, Ricardo Enrique Grados-Torrez 2, Pablo Leivar4, Meritxell Antolín-Llovera5,Carmen López-Iglesias, Nuria Cortadellas, Joan Carles Ferrer, and Narciso Campos*

Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, 08028Barcelona, Spain (S.F., P.L., M.A.-L., J.C.F., N.Ca.); Departament de Genètica Molecular, Centre de Recerca enAgrigenòmica (Consejo Superior de Investigaciones Científicas-Institut de Recerca i TecnologiaAgroalimentàries-Universitat Autònoma de Barcelona-Universitat de Barcelona), Bellaterra (Cerdanyola delVallès), 08193 Barcelona, Spain (R.E.G.-T., N.Ca.); Crio-Microscopia Electrònica, Centres Científics iTecnològics, Campus Pedralbes, Universitat de Barcelona, 08028 Barcelona, Spain (C.L.-I.); and Unitat deMicroscopia Electrònica, Centres Científics i Tecnològics, Campus Casanova, Universitat de Barcelona, 08036Barcelona, Spain (N.Co.)

ORCID IDs: 0000-0003-4878-3684 (P.L.); 0000-0002-9685-0549 (N. Campos).

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) has a key regulatory role in the mevalonate pathwayfor isoprenoid biosynthesis and is composed of an endoplasmic reticulum (ER)-anchoring membrane domain with low sequencesimilarity among eukaryotic kingdoms and a conserved cytosolic catalytic domain. Organized smooth endoplasmic reticulum(OSER) structures are common formations of hypertrophied tightly packed ER membranes devoted to specific biosynthetic andsecretory functions, the biogenesis of which remains largely unexplored. We show that the membrane domain of plant HMGRsuffices to trigger ER proliferation and OSER biogenesis. The proliferating membranes become highly enriched in HMGRprotein, but they do not accumulate sterols, indicating a morphogenetic rather than a metabolic role for HMGR. TheN-terminal MDVRRRPP motif present in most plant HMGR isoforms is not required for retention in the ER, which waspreviously proposed, but functions as an ER morphogenic signal. Plant OSER structures are morphologically similar to thoseof animal cells, emerge from tripartite ER junctions, and mainly build up beside the nuclear envelope, indicating conservedOSER biogenesis in high eukaryotes. Factors other than the OSER-inducing HMGR construct mediate the tight apposition of theproliferating membranes, implying separate ER proliferation and membrane association steps. Overexpression of the membranedomain of Arabidopsis (Arabidopsis thaliana) HMGR leads to ER hypertrophy in every tested cell type and plant species, whereasthe knockout of the HMG1 gene from Arabidopsis, encoding its major HMGR isoform, causes ER aggregation at the nuclearenvelope. Our results show that the membrane domain of HMGR contributes to ER morphogenesis in plant cells.

The endoplasmic reticulum (ER) consists of a cytosolicdynamic weft of tubules and cisternae, with varyinggeneral shape and extension, continuously adapted tothe changing demands of its functional commitment(Staehelin, 1997; Federovitch et al., 2005; Stefano et al.,2014). A high increase in the number and extension of ERmembranes (ER hypertrophy) is a common feature ofeukaryotic cells specialized in biosynthesis and secretionof metabolites and proteins. This is, for instance, the caseof plant gland cells involved in the production of diverseisoprenoid products (Schnepf, 1974) and mammalianadrenocortical cells involved in steroid hormone biosyn-thesis (Fawcett, 1981). Such processes require elevatedlevels of ER resident enzymes like 3-hydroxy-3-methylglutaryl CoA reductase (HMGR), cytochrome b5,and cytochrome P450 (Boutanaev et al., 2015; Storbecket al., 2014). Interestingly, the overexpression of theseenzymes in yeast (Saccharomyces cerevisiae) or mammalian

cells induces proliferation and accumulation of ERmembranes that, at the electron microscope, are visual-ized as stacked membranes (lamellae or karmellae), con-centric membrane associations (whorled ER), or orderedarrays of membranes with hexagonal or cubic symmetry(crystalloid ER; Jingami et al., 1987; Wright et al., 1988).These structures have been collectively named organizedsmooth endoplasmic reticulum (OSER; Snapp et al.,2003). OSER structures are natural formations broadlydistributed in animal and plant cells (Snapp, 2005;Almsherqi et al., 2009) and can be induced in yeast andmammalian cells by overexpression of about a dozenendogenous ERmembrane proteins (Sandig et al., 1999).Overexpression studies led to the proposal that OSERstructures are caused by membrane zippering becauseof oligomerization of the expressed protein (Takei et al.,1994; Gong et al., 1996; Yamamoto et al., 1996; Fukudaet al., 2001; Li et al., 2003; Snapp et al., 2003; Lingwood

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et al., 2009; Costantini et al., 2012), but other biochemicalprocesses are likely involved in OSER biogenesis. Thehuge membrane proliferation observed upon expressionof OSER-inducing proteins, such as yeast or humanHMGR (Jingami et al., 1987; Wright et al., 1988) or di-verse cytochrome P450 variants (Sandig et al., 1999),requires very active lipid and protein biosynthesis andcannot be explained by mere oligomerization. Despitelong-lasting research, the mechanisms and signals trig-gering ER proliferation and OSER biogenesis remainelusive (Wright, 1993; Federovitch et al., 2005). Particularattention has been paid to identification of sequence orstructural features in the overexpressed protein criticalfor its OSER-inducing effect (Jingami et al., 1987;Vergères et al., 1993; Parrish et al., 1995; Yamamoto et al.,1996; Profant et al., 1999, 2000). However, the work todate has not allowed the identification of broadly con-served peptide or structural motifs that function as asignal to trigger ER proliferation and morphogenesis.

Eukaryotic HMGR has a key regulatory role in themevalonate pathway for isoprenoid biosynthesis (Burg

and Espenshade, 2011; Rodríguez-Concepción et al.,2013). Isoprenoid products derived from the pathway areinvolved in diverse essential functions, including mem-brane biogenesis (sterols), control of growth and devel-opment (steroid hormones and cytokinins), proteinprenylation (farnesyl and geranyl groups), protein gly-cosylation (dolichols), and respiration (ubiquinones). Inplants, the mevalonate pathway also provides a widevariety of compounds required for defense or positiveinteractions with other organisms (Chappell, 1995).HMGR has a bipartite structure formed by an N-terminalmembrane domain, with low sequence similarity amongeukaryotic kingdoms, and a highly conserved catalyticdomain, and the two are connected by a flexible non-conserved linker region (Liscum et al., 1985; Basson et al.,1988; Campos and Boronat, 1995). HMGR is tetrameric inanimals and plants because of oligomerization of thecatalytic domain (Bach et al., 1986; Wititsuwannakulet al., 1990; Istvan et al., 2000). The membrane domain ofplant HMGR, which is composed of two conserved hy-drophobic segments, a short lumenal loop and a cytosolicN-terminal region (Campos and Boronat, 1995), is fairlysmall compared with those of yeast and human HMGR,which are predicted to have eight transmembrane se-quences (Olender and Simon, 1992; Profant et al., 2000).The N-terminal region is diverged among plant HMGRisoforms but contains a highly conserved N-terminal Argmotif that fits the consensus of ER retention signal de-scribed in animal type II membrane proteins (Camposand Boronat, 1995). Despite their structural differences, alleukaryotic HMGRs coincide in basic functional features.The membrane domains of yeast and human HMGRdetermine the targeting, turnover, and capacity to induceER proliferation, therefore controlling the catalytic activ-ity of the appended domain (Gil et al., 1985; Jingami et al.,1987; Skalnik et al., 1988; Olender and Simon, 1992;Parrish et al., 1995; Hampton et al., 1996). Conversely,a properly folded cytosolic domain is required to triggerkarmellae biogenesis in yeast or slow down regulatedHMGR degradation in human cells, indicating that thetertiary structure and function of the membrane domainare influenced by the oligomerization state of the catalyticdomain (Cheng et al., 1999; Profant et al., 1999). Thus, inyeast and animal cells, membrane and catalytic HMGRdomains show interdependent function.

In Arabidopsis (Arabidopsis thaliana), two genes en-code three HMGR isoforms: HMGR1S and HMGR1Lare encoded by HMG1, and HMGR2 is encoded byHMG2 (Enjuto et al., 1994; Lumbreras et al., 1995). Iso-zymes HMGR1S and HMGR1L are identical in se-quence, but HMGR1L has an N-terminal extension of50-amino acid residues (1L extra region). HMGR1Stranscript is expressed in the whole plant, whereas thoseencoding HMGR2 or HMGR1L are detected at a muchlower level and only in meristems, seedlings, roots, andinflorescences (Enjuto et al., 1994, 1995; Lumbreras et al.,1995). Chimeras formed by the membrane domain ofplant HMGR fused to GFP localize in the ER networkbut accumulate in highly fluorescent cytosolic bodies(Leivar et al., 2005; Merret et al., 2007). In this work,

1 This work was supported by the Ministerio de Ciencia y Tec-nología (predoctoral scholarship to S.F.), the Ministerio deAsuntos Exteriores y de Cooperación-Agencia Española deCooperación Internacional para el Desarrollo (predoctoral schol-arship to R.E.G.-T.), the Comissió Interdepartamental de Recerca iInnovació Tecnològica (predoctoral scholarship to P.L.), the Min-isterio de Educación, Cultura y Deporte (predoctoral scholarshipto M.A.-L.), the Spanish Government (grant nos. BMC2003–03450to N.Ca., BFU2006–14655 to N.Ca., BFU2011–24208 to N.Ca., andCSD2007–00036 to Centre de Recerca en Agrigenòmica), FondoEuropeo de Desarrollo Regional, and the Generalitat de Catalunya(grant no. 2014SGR1434).

2 These authors contributed equally to the article.3 Present address: Dipartimento di Scienze Biomolecolari e Biotec-

nologie, Università degli Studi di Milano, 20133 Milan, Italy.4 Present address: Departament de Bioenginyeria, Institut Químic

de Sarrià, Universitat Ramon Llull, 08017 Barcelona, Spain.5 Present address: Genetics, Department of Biology, University of

Munich, 82152 Munich-Martinsried, Germany.* Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Narciso Campos ([email protected]).

S.F. prepared and characterized the 1S:GFP transgenic plants,identified the morphogenic properties of 1S:GFP monomeric, andperformed the whole-mount analysis; R.E.G.-T. finished the trans-genic plant characterization and whole-mount analysis and per-formed confocal microscopy and electron microscopy studies inNicotiana benthamiana and Arabidopsis (Arabidopsis thaliana); S.F.and R.E.G.-T. carried out most of the experiments and contributedequally to the work; R.E.G.-T., M.A.-L., and N.Ca. characterized the3-hydroxy-3-methylglutaryl CoA reductase mutant; P.L. and M.A.-L.did the starting characterization of 3-hydroxy-3-methylglutaryl CoAreductase:GFP chimeras; C.L.-I., N.Co., and J.C.F. assisted with themicroscopy studies; N.Ca. conceived and directed the project, con-tributed to the microscopy and molecular biology studies, did thebioinformatic analysis, and wrote the article; All authors discussedthe results and commented on the article.

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we show that these bodies are OSER structures. Expres-sion of the membrane domain of HMGR1S or HMGR2fused to GFP leads to reversible formation of OSERstructures in every cell type of diverse plant species. Theconserved N-terminal Arg motif is not an ER retentionsignal but is required to induce ER proliferation andmorphogenesis. In addition, the knock of theHMG1 genefrom Arabidopsis causes severe defects in ER morphol-ogy around the nucleus. These results indicate that themembrane domain of plant HMGR is naturally involvedin the morphogenesis of the ER.

RESULTS

1S:GFP and 2:GFP Induce Biogenesis of ER Aggregates,But 1L:GFP Is Retained in the ER Network

Plant HMGR is primarily targeted to the ER by inter-action of the highly conserved hydrophobic sequences ofits membrane domain with the signal recognition particle(Campos and Boronat, 1995). To find out whether the lessconserved regions flanking these sequences might influ-ence the final subcellular location, we designed chimerasof the three Arabidopsis HMGR isoforms carrying thefluorescent marker GFP instead of the catalytic domain(Fig. 1A). Chimeras 1S:GFP, 1L:GFP, and 2:GFP containthe membrane domains of Arabidopsis HMGR1S,HMGR1L, and HMGR2, respectively. The chimeras weretransiently expressed by biolistic transfection or agro-infection in different cell types fromArabidopsis,Nicotianabenthamiana, onion (Allium cepa), and leek (Allium porrum).In epidermal, vascular, mesophyll, trichome, and culturecells from Arabidopsis and epidermal cells from onionand leek, 1S:GFP and 2:GFP are detected in the ERnetwork but mainly accumulate in highly fluorescent

vesicular structures, which remain close to the ER (Fig.1, B–E; Supplemental Figs. S1, A–D, S2, A–C, and S3,A–F). In all assayed systems, these structures emerge assmall spherical bodies in three-branch junctions of thecortical ER or beside the nuclear envelope (Fig. 1, B–D;Supplemental Figs. S2, A–C and S3, A–F) and remainphysically connected to the ER network, which wasconfirmed by three-dimensional reconstruction of con-focal microscopy images (Supplemental Movie S1).They can become large fluorescent bodies usuallylocated near the nucleus (Fig. 1D). In transfected N.benthamiana leaves, 1S:GFPwas expressed at high levels inepidermal cells and mostly accumulated in large,densely packed structures around the nucleus (Fig. 1G).In parallel transfections of Arabidopsis, N. benthamiana,and onion, the ER lumen markers GFP-Lys-Asp-Glu-Leu (GFP-KDEL) and GFP-His-Asp-Glu-Leu (GFP-HDEL) outlined an ER network characteristic of thetargeted cell type (Fig. 1I; Supplemental Figs. S1E, S2E,and S3I), confirming that the aggregates were formedde novo upon 1S:GFP or 2:GFP expression. In contrastto 1S:GFP and 2:GFP, the 1L:GFP chimera was exclu-sively found in the ER network in all transfected plantsystems, which included epidermal cells from Arabi-dopsis, N. benthamiana, and onion and an undifferen-tiated cell line from Arabidopsis (Fig. 1, F and H;Supplemental Figs. S2D and S3, G and H). Because1S:GFP and 1L:GFP behave differently, formation of theER-derived aggregates is likely prevented by the 1Lextra region.

The physical connection of the 1S:GFP-containingaggregates to the ER network suggested but did notprove that they are part of the ER. To examine theiridentity, we cotransfected 1S:GFP with T3RE, whichis a Discosoma spp. red fluorescent protein derivative

Figure 1. ER morphology after expressionof 1S:GFP, 2:GFP, and 1L:GFP in Arabi-dopsis and N. benthamiana cells. A, Sche-matic representation in the ER membrane ofHMGR:GFP chimeras used in this study.Chimeras 1S:GFP, 1L:GFP, and 2:GFP havethe membrane domains of ArabidopsisHMGR1S, HMGR1L, and HMGR2, respec-tively, fused to C-terminal GFP. Construct 1Sis in the membrane domain of HMGR1Salone. B to L, Arabidopsis (B–F) andN. benthamiana (G–L) leaves were transfectedwith constructs encoding the HMGR chi-mera (1S:GFP, 2:GFP, or 1L:GFP) or a controlER lumen marker (GFP-HDEL or T3RE) andvisualized by confocal laser microscopy. J toL, Cotransfection with 1S:GFPand T3RE wasvisualized by confocal laser microscopy asindicated. The images are Z projections of10 (B–F), 50 (G–I), or 20 (J–L) sections fromtransfected Arabidopsis epidermal (B, C, andF), Arabidopsis vascular (D and E), or N.benthamiana epidermal (G–L) cells. N, Nu-cleus. Bars = 10 mm.

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targeted to the ER lumen (Forés et al., 2006). In N.benthamiana, Arabidopsis, and onion epidermal cells, therewas a perfect match between 1S:GFP and T3RE fluo-rescence in the ER network, nuclear envelope, andspherical structures (Fig. 1, J–L; Supplemental Fig. S4).Because T3RE carries a functional ER retention motif(C-terminal KDEL), the physical coincidence impliesthat the 1S:GFP-containing aggregates belong to the ER.The 1S:GFP chimera can be used as a general marker ofthe altered ER: it labels all ER domains, including theinduced ER membrane aggregates.

The Biogenesis of ER Aggregates Induced by 1S:GFPIs Reversible

The 1S:GFP chimera was assayed also by stabletransformation of Arabidopsis plants. Six independenttransgenic lines expressing high levels of 1S:GFP in thewhole plant (Fig. 2A) were brought to homozygosityto prevent segregation in progeny. Alteration of the ERmorphology was general in 7-d-old 1S:GFP plants. ER-derived vesicular structures were observed, for in-stance, in cotyledon epidermal cells, root hairs, andstomata guard cells (Fig. 2, B–D). Although the trans-genic lines had a slightly delayed growth with respectto the wild type, they were phenotypically normal. Theobserved disturbance of the ER structure did not blockER function and plant development. As in the tran-sient expression assays, 1S:GFP aggregates developed

from ER network junctions (Fig. 2E) and attained thelargest size at the nuclear envelope (Fig. 2, B–D).Interestingly, 1S:GFP expression vanished betweenweeks 3 and 4 of plant development (Fig. 2F) but re-producibly reappeared upon seed germination, at leastuntil generation five. The ER was altered in cells stillexpressing 1S:GFP (Fig. 2G) but had normal networkstructure in cells without the chimera, which was in-dicated by transient expression of lumenal ER markers(Fig. 2, H and I). Biolistic transfection with the ERmarker was conducted shortly after disappearance of1S:GFP, ruling out that the observed network couldbelong to a newly differentiated cell. The transfectedleaves had the expected anatomy, with no signs ofplant tissue degeneration or regeneration (Fig. 2F). Weconclude that the loss of 1S:GFP allowed progressiverecovery of normal ER architecture, which was alteredagain in the next generation, because of reappearanceof the chimera. ER disturbance driven by 1S:GFP doesnot irreversibly affect the organelle and is compatiblewith cell integrity and function.

The 1S:GFP-Induced ER Aggregates Are OSER StructuresThat Result from Membrane Proliferation

To further characterize the ER-derived vesicularstructures, we analyzed N. benthamiana and Arabi-dopsis 1S:GFP-expressing organs by transmission elec-tron microscopy (EM). In N. benthamiana epidermal

Figure 2. Reversible formation of ER aggregatesin transgenic 1S:GFP Arabidopsis plants. Trans-genic plants were analyzed by stereomicroscopy(A and F) and confocal laser microscopy (B–E andG–I) to detect the fluorescence of 1S:GFP (green;A–G), endogenous chlorophyll (red; F), or ERlumen markers (red or green; H and I). A, Ten-day-old transgenic seedlings have widespread 1S:GFP expression. B to D, The chimera induces ER-derived aggregates in all cell types, including leafepidermal cells (B and E), root hairs (C), andstomata guard cells (D). F, After 3 weeks ofgrowth, 1S:GFP vanishes, allowing detection ofred fluorescence from chlorophyll. G to I, ER-derived aggregates are present in leaf sectors stillexpressing 1S:GFP (G) but not in areas withoutthe chimera, which is shown by biolistic trans-fection of ER lumen markers (H and I). Confocalmicroscope images are Z projections of 12 (B, D,E, H, and I) and 21 (G) sections from the trans-fected cells, except in C, which corresponds to asingle section. N, Nucleus. Bars = 3 mm (A and F)and 10 mm (B to E and G to I).


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cells, 1S:GFP expression correlates with the appearanceof densely packed ER membrane aggregates (Fig. 3, Aand B), which were never seen in nontransfected or-gans. The hypertrophied ER had ordered membraneswith crystalloid, lamellar, or whorled patterns (Fig. 3B),

such as the OSER structures previously described inanimal cells (Snapp et al., 2003). Also coincident withthem, 1S:GFP-induced OSER structures have a narrowcytosolic space of about 10 nm between membranes,whereas the broader areas correspond to the ER lumen

Figure 3. Ultrastructural analysis of 1S:GFP-induced ER aggregates. Agroinfected N. benthamiana leaves (A–E) and transgenicArabidopsis seedlings (F–I) expressing 1S:GFP were imaged by transmission EM. A to E, In N. benthamiana leaf epidermal cells,the aggregates are composed of interconnected crystalloid, lamellar, and whorled smooth ER (A and B) frequently locatedbeside the nucleus (C). Whorled structures contain alternating lumenal (aligned dots) and more electron-dense cytosolic spacesthat exclude ribosomes (white arrowheads; D). The narrow (10 nm) electron-dense space between membranes (arrowhead)shows continuity with the cytosol (E). F to I, In all cell types of transgenic 1S:GFP Arabidopsis seedlings, the ER membraneaggregates also accumulate around the nucleus and have diverse interconnected patterns. Cr, Crystalloid; La, lamellar; N,nucleus; n, nucleolus; W, whorled. Bars = 2 mm (A), 0.5 mm (B), 1 mm (C and F), 200 nm (D), 100 nm (E), and 0.5 mm (G–I).





(Fig. 3, D and E). Ribosomes are excluded from theaggregates, which only contain smooth ER (Fig. 3, B, D,and E). Similar smooth ER patterns with narrow cyto-solic space were observed in all organs and cell types oftransgenic 1S:GFP Arabidopsis plants (Fig. 3, F–I). ERregions with crystalloid, whorled, or lamellar mem-branes were interconnected in the same aggregate, in-dicating that the pattern may depend on the orientationof the section (Fig. 3, B, D, and F–I). The crystalloidstructures of both Arabidopsis and N. benthamiana cellsfrequently appear beside the nuclear envelope (Fig. 3, Cand F), which is in agreement with the observations atthe confocal microscope. Ultrastructural and confocalmicroscope analyses indicate that expression of 1S:GFPor 2:GFP leads to a high increase of the amount of ERmembranes. The 1S:GFP and 2:GFP chimeras are veryeffective inducers of membrane proliferation and OSERbiogenesis in plant cells.

The OSER Structures Have High Levels of 1S:GFP andHMGR But Low Sterol Content

To confirm the identity of 1S:GFP-derived OSER struc-tures and determine whether they also contain endoge-nous HMGR, we performed double-immunocytochemicalanalyses on transgenic 1S:GFP Arabidopsis plants, withpolyclonal antibodies raised against GFP or the catalyticdomain of HMGR1. For a panoramic view of single cells,we first ran whole-mount immunocytochemistry on 6-d-old seedlings. In cotyledon parenchymal cells, signalscorresponding to 1S:GFP and endogenous HMGR (fluo-rescing in green and red, respectively) physically coin-cided in an irregular mass of ER around the blue-labelednucleus (Fig. 4, A–C). The ER was concentrated sur-rounding this organelle, with poor cortical ER (Fig. 4A).

The subcellular locations of 1S:GFP and endogenousHMGR were further analyzed by immunocytochemicalultrastructural studies. In leaf epidermal cells of thetransgenic Arabidopsis seedlings, 1S:GFP and endoge-nous HMGR were detected at high densities on mem-branes of ER-derived aggregates (Fig. 4, D and E). Mixeddual labeling was observed in crystalloid, whorled, andlamellar ER aggregates, the interconnecting ER network,and the nuclear envelope (Fig. 4, E–G). No signal wasdetected in the control without primary antibody (Fig.4H) or controls with primary and secondary antibodiesin other cell compartments (Supplemental Fig. S5). Theobservation of normal morphologies for the Golgi,plasmalemma, mitochondria, and chloroplasts indicatesthat membrane proliferation induced by 1S:GFP is ex-clusive of the ER (Supplemental Fig. S5). Our resultsindicate that 1S:GFP and endogenous HMGR colocalizein all regions of the ER but accumulate mainly in the ER-derived aggregates, which we refer to as ER-HMGRdomains. The immunodetection of GFP shows that thesedomains correspond to the highly fluorescent bodiesidentified in the confocal microscope. There is full coin-cidence in subcellular location between 1S:GFP and en-dogenous HMGR in both confocal microscopy and EM,

indicating that the elimination of the catalytic domain doesnot alter the subcellular distribution of the HMGR protein,which is entirely determined by its membrane domain.

The high abundance of endogenous HMGR in 1S:GFP-induced OSER structures suggested that HMGRaccumulates at higher levels in transgenic plants than inwild-type plants. To examine this hypothesis, crude ex-tracts from Arabidopsis seedlings were submitted toimmunoblot with an antibody specific for the HMGRcatalytic domain. As shown in Figure 4I, the HMGRprotein level was about 3 times higher in transgenic1S:GFP plants than in the wild type. To ascertain whetherthis increase was a consequence of elevated transcriptlevels, we ran quantitative reverse transcription and real-time PCR with separate primer sets specific for HMG1and HMG2 (Fig. 4I). We found that the total HMGRtranscript did not increase but consistently decreased inthe transgenic 1S:GFP plants. Thus, the increase inHMGR protein was not caused by higher transcriptlevels. The 1S:GFP chimera and endogenous HMGRaccumulate in OSER structures induced by the mem-brane domain of HMGR1S, which has a self-stabilizationeffect. To find out whether the higher HMGR levelcaused sterol accumulation, we submitted roots of thetransgenic plants to sterol-specific staining with filipin III(Boutté et al., 2011). Although the filipin signal was ev-ident in the plasmalemma and some intracellular struc-tures, it was nearly undetectable in 1S:GFP-inducedOSER structures (Fig. 4, J–L), indicating very low levelsof sterols with the unreacted 39-b-OH group. The mainrole of plant HMGR in OSER biogenesis does not seemto be metabolic but morphogenetic.

The Membrane Domain of HMGR1S Suffices to TriggerOSER Biogenesis

It was previously shown that expression of dimeriz-ing GFP fused to the cytoplasmic domain of otherwisepassive ER resident proteins causes ER membranestacking and OSER biogenesis and that equivalent con-structs with a monomeric GFP variant do not have thismorphogenic effect (Snapp et al., 2003; Costantini et al.,2012). To find out whether dimerization of GFP wascontributing to the OSER-inducing capacity of 1S:GFPin plant cells, we designed the 1S:GFP monomeric(1S:GFPm) chimera containing a nondimerizing GFP. Ofthe three single-amino acid changes known to preventGFP dimerization (Zacharias et al., 2002), we chose theA206K substitution, because it has the lowest dimer-ization capacity. Its dissociation constant is about 650times higher than that of the original GFP, making thecorresponding dimer essentially undetectable (Zachariaset al., 2002). The morphogenic capacity of 1S:GFPm wasfirst analyzed by stable transformation of the Arabi-dopsis SALK_061790 knockout mutant (Myouga et al.,2010), which does not express HMGR1S or HMGR1L. InSALK_061790 parenchymal cells, 1S:GFPm expressionconverted the ER network into perinuclear and corticalER aggregates containing the monomeric chimera, which


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Figure 4. 1S:GFP-induced OSER structures have high levels of 1S:GFPand HMGR but low sterol content. A to C, Whole-mountimmunocytochemical analysis of transgenic 1S:GFP Arabidopsis seedlings. Immunodetection of 1S:GFP (green) and HMGR(red) in cotyledon parenchymal cells visualized by confocal microscopy. The two proteins colocalize in ER aggregates aroundthe DAPI-stained (blue) nucleus. D to H, Immunocytochemical EM analysis of transgenic 1S:GFP Arabidopsis seedlings. 1S:GFP(18-nm particle) and endogenous HMGR (12-nm particle) were immunodetected in leaf epidermal cells. The two proteinscolocalize on membranes of crystalloid aggregates (D, E, and G), whorled aggregates (F), and nuclear envelope (arrowhead in





was shown by whole-mount immunocytochemical anal-ysis (Fig. 5A). No endogenous HMGR could be detectedunder the same conditions (Fig. 5B), confirming that theobserved alterations were not caused by a sharp increasein HMG2 expression. In leaf epidermal cells from wild-type Arabidopsis, transient expression of 1S:GFPm led tothe emergence of ER-derived vesicles from ER junctions(Fig. 5C), similar to those induced by transient or stableexpression of 1S:GFP (Figs. 1B and 2E). The 1S:GFPmaggregates became as large as those induced by 1S:GFP(compare Fig. 5D with Fig. 1D). The 1S:GFPm chimeraaccumulated as well in thick ER strands that were con-nected to the thin ER network (Fig. 5E). In N. benthamianaepidermal cells, 1S:GFPm led to formation of big perinu-clear ER aggregates with radially disposed thick ER strands(Fig. 5F). Thus, the appearance of thick ER strands is reit-erative in cells from different plants expressing 1S:GFPm.

To confirm that the membrane domain of HMGR1S isself-sufficient to induce OSER structures, we removedGFP from the 1S:GFP chimera. This was achieved byincorporation of two consecutive stop codons at an ap-propriate site of the 1S:GFP coding sequence withoutaltering other sequences in the expression plasmid. Theresulting construct encoded a 20-kD HMGR1S fragment,named 1S, containing the N-terminal region, the twomembrane-spanning segments, and the linker region. InN. benthamiana leaf epidermal cells, the nonfluorescent1S fragment led to formation of ER aggregates, whichwere visualized by cotransfected T3RE, whereas whenexpressed alone, the ER lumen marker localized in thetypical ER network (Fig. 5, G and H). This indicates thatthe membrane domain of HMGR1S devoid of anappended cytosolic domain can disturb ER morphologyand induce OSER structures. ER proliferation and OSERbiogenesis were not as pronounced as those induced by1S:GFP or 1S:GFPm chimeras, probably because oflower expression or stability of the 1S fragment.

To characterize OSER structures produced in theabsence of dimerizing GFP, we obtained electron mi-crographs of agroinfected N. benthamiana leaves. Ultra-structural analysis of the epidermal cells uncovered thatexpression of 1S:GFPm led to very active proliferationof the smooth ER that was converted to interconnectedcrystalloid, lamellar, and whorled aggregates, equivalentto those generated by 1S:GFP (Fig. 5, I–L). Particular ul-trastructural features, such as exclusion of ribosomesfrom the aggregate and the narrow and regular cytosolicspaces between membranes (Fig. 5, K and L), also coin-cided with those of 1S:GFP-induced OSER formations.

The apposition of the cytosolic side of the ER membranesis remarkable, because monomeric GFP cannot contributeto the close interaction. Most likely, other ER residentproteins accomplished this task. We conclude that ex-pression of 1S:GFPm in plant cells leads to ER prolifera-tion and association of the accumulated membranesrendering typical OSER structures.

The Conserved N-Terminal Motif of Plant HMGR Is anOSER-Inducing Signal

It was previously suggested that the Arg motif con-served in the N-terminal region of plant HMGRmight bean ER retention signal (Campos and Boronat, 1995;Merret et al., 2007). As a first step to examine its function,we searched for all available N-terminal plant HMGR-encoding sequences in the National Center for Biotech-nology Information database. To avoid overestimatingvariability, only full-length complementary DNAs andESTs confirmed by at least two independent entries wereconsidered. We found 305 nonredundant HMGR se-quences from 158 species and 117 genera of mono-tyledonae and dicotyledonae, which broadly representthe flowering plant kingdom (Supplemental Data Set S1).In these sequences, the three-Arg tag is only part of alonger motif consisting of the N-terminal Met, one or twoacidic residues, a hydrophobic residue, the three argi-nines, and two prolines (Fig. 6A); 273 of 305 plantHMGR isoforms (90%) contain this canonical sequence(Supplemental Data Set S1). From them, 194 sequenceskeep all three arginines, whereas 79 have introduced one-or two-amino acid substitutions. The most repeated var-iations of the three-Arg tag (written in one-letter code) areHRR (17 sequences), RQR (9 sequences), SRR (8 sequences),RRK (7 sequences), RRL (4 sequences), RRQ (4 sequences),HRK (3 sequences), and RRG (3 sequences). These variantskeep at least two arginines or three basic residues, indi-cating sequence constraints for a conserved function.

To examine the role of the conserved N-terminal motif,we obtained new variants of 1S:GFP, in which theDLRRR sequence was replaced by a single Gly residue(construct DDLRRR-1S:GFP) or the three arginines werereplaced by three alanines (construct DRRR-1S:GFP; Fig.6). The chimeras were transiently expressed in leaf andcell culture from Arabidopsis or N. benthamiana. In epi-dermal, vascular, or in vitro cultured cells, both mutatedvariants localized in the cortical ER network (Fig. 6, B,D, F, and H). No OSER structures were induced byDDLRRR-1S:GFP or DRRR-1S:GFP, despite the long-lasting

Figure 4. (Continued.)G). No signal is observed in the control without primary antibodies (H). I, Quantification of HMG1 and HMG2 transcripts andHMGR protein in transgenic 1S:GFP seedlings. Transcript and protein levels were measured by qRT-PCR or immunoblot and arerepresented relative to the HMG1 transcript or HMGR1S protein of wild-type seedlings, respectively, as the average 6 SEM of threeindependent experiments. Electrophoretic migration of HMGR1L (69 kD) and HMGR1S (63 kD) is indicated. Coomassie-stainedRubisco was used as a loading control and is shown below the immunoblot. J to K, Sterol and 1S:GFP distribution in root corticalcells in transgenic 1S:GFP seedlings. Samples were stained with filipin III and visualized by confocal microscopy. Free sterols showpoor colocalization with 1S:GFP. Confocal microscope images are Z projections of 10 (A–C) or 15 (J–L) sections. Cr, Crystalloid; N,nucleus; W, whorled; wt, wild type. Bars = 10 mm (A–C), 1 mm (D), 200 nm (E and F), 0.5 mm (G and H), and 30 mm (J–L).


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expression, which depending of the system, was pro-longed for 18 to 96 h. Under the same conditions, the1S:GFP chimera induced typical OSER structures (Fig. 6,C, E, G, and I). We conclude that the conserved Arg motifis required for ER proliferation and OSER biogenesistriggered by 1S:GFP but not for retention in the ER, whichshould be mediated by other elements of the N-terminaldomain. In plant HMGR, the conserved N-terminal motifsignals ER proliferation and morphogenesis.

In Arabidopsis, the Disruption of the HMG1 Gene CausesER Aggregation around the Nucleus

The requirement of a highly conserved HMGR motiffor OSER biogenesis in plant cells and the OSER-inducing

capacity of this protein in the three eukaryotic kingdomsstrongly suggested that HMGR has a natural role inthe morphogenesis of the ER. To further examine thishypothesis, we determined ER morphology in theSALK_061790 line from Arabidopsis, which carries atransfer DNA (T-DNA) insertion in the HMG1 gene andis the knockout for HMGR1S and HMGR1L. Wetransformed this line with GFP-HDEL to trace the ERlumen. Transformation was performed in heterozygousSALK_061790 plants, and the morphology of the ERwas studied in generation T2, which had a segregatingT-DNA insertion genotype. This allowed comparison ofthe three HMG1 allelic combinations on the same ge-netic background. The ER was characterized in cellsfrom the root cortex of 7- to 10-d-old plants. At this age,the homozygous mutant was at the two-cotyledon stage

Figure 5. OSER structures induced by 1S:GFPm and 1S fragment. A and B, Whole-mount immunocytochemical analysis of theSALK_061790 Arabidopsis mutant stably transformed with 1S:GFPm. Immunodetection of 1S:GFPm (A) and HMGR (B) in coty-ledon parenchymal cells of 1S:GFPm/SALK_061790 seedlings visualized by confocal microscopy with DAPI-stained nuclei. C to F,Transient expression of 1S:GFPm in epidermal cells from wild-type Arabidopsis (C–E) and N. benthamiana (F) leaves visualized byconfocal microscopy. G and H, Transient coexpression of 1S fragment and T3RE ER marker (G) in epidermal cells fromN. benthamiana leaves using expression of T3RE alone as a control (H). I to L, Transmission EM of epidermal cells fromN. benthamiana leaf after transient expression of 1S:GFPm. Confocal microscope images are Z projections of 10 (A–E), 33 (F), 15(G), and 19 (H) sections. Cr, Crystalloid; N, nucleus; W, whorled. Bars = 10 mm (A–H), 1 mm (I), 0.5 mm (J and K), and 200 nm (L).





and could be easily distinguished from homozygouswild-type and heterozygous plants, which already hadtrue leaves (Fig. 7H). We examined the nuclear andcortical ER in root cells from 36 mutant and 55 wild-type plants. In total, 406 Z-projection images wereobtained (201 from the mutant and 205 from the wildtype) with reproducible ER morphologies. In the cortexfrom homozygous wild-type and heterozygous plants,the GFP-HDEL labeling was prominent in ER bodiesand weaker in the cortical ER network and the nuclearenvelope (Fig. 7, A–C). The ER bodies are natural fu-siform enlargements of the ER and very abundant inArabidopsis seedlings (Yamada et al., 2011). In the wildtype, some ER bodies were around the nucleus but onlyoccasionally in contact with the nuclear envelope (Fig.7A). However, in homozygous mutant cells, ER bodiesaggregated at the nuclear envelope (Fig. 7, D–F). Underbright field, the nucleus and surrounding ER bod-ies formed a clearly visible rough mass (Fig. 7F),which could be identified as well by 49,6-diamidino-2-phenylindole (DAPI) staining (Fig. 7J). The differencesbetween the wild type and the mutant are more evi-dent in Z-projection images of single nuclei (Fig. 7, Gand I) and videos generated from the correspondingindividual sections (Supplemental Movies S2 and S3).A cross optical section of the root confirmed that thenuclear aggregates of ER bodies occurred in the cortex(just below the epidermal layer; Fig. 7, K and L). Thedisruption of the HMG1 gene caused concentration ofER bodies at the nuclear envelope but did not affect thecortical ER network and the ER bodies connected to it,which was observed in optical section series (compareSupplemental Movie S2 with Supplemental Movie S3).We conclude that the knockout of HMG1 gene fromArabidopsis selectively affects the organization of theER around the nucleus, causing aggregation of ERbodies.

DISCUSSION

We have shown that the membrane domain of Ara-bidopsis HMGR is involved in the morphogenesis of theER. On the one hand, the knockout of the HMG1 genefrom Arabidopsis encoding its major HMGR isoform al-tered the ER morphology around the nucleus. On theother hand, the membrane domain of HMGR1S orHMGR2 fused to GFP-induced massive ER proliferationand OSER biogenesis through a specific well-conservedsequence. The OSER structures emerged from ER junc-tions and mostly accumulated around the nucleus. Sim-ilar alterations were induced by 1S:GFP and 2:GFP in alltested cell types from diverse plant species, indicatinga conserved mechanism. The nondimerizing 1S:GFPmchimera was as effective as 1S:GFP in the induction of ERproliferation and membrane stacking, indicating that anappended dimerizing cytosolic domain is not requiredfor the morphogenic effect. Expression of the membranedomain of HMGR1S alone also disturbed the ER networkand generated OSER structures, confirming its sufficiency

Figure 6. Role of the N-terminal motif of plant HMGR in the mor-phogenesis of the ER. A, N-terminal motif of plant HMGR de-duced from nonredundant complementary DNA and EST entries(Supplemental Data Set S1) with MEME software (Bailey et al., 2009). Lettersize is proportional to amino acid residue frequency in the referred position.The amount of motif-defining information corresponding to each amino acidposition is represented in the ordinate, in bits. B to I, The original 1S:GFP(N-terminal sequence MDLRRRPPKPP) and its mutated variants DDLRRR-1S:GFP (sequence MGPPKPP) and DRRR-1S:GFP (sequence MDLAAAPPKPP)were transiently expressed by biolistic transfection in Arabidopsis leaf (B–E) orby agroinfection in Arabidopsis cell culture (F and G) and N. benthamianaleaf (H and I). Vascular (B and C), epidermal (D, E, H, and I), and in vitroculture cells (F and G) were visualized by confocal microscopy. Images areZ projections of 10 (B–G) or 50 (H and I) sections. N, Nucleus. Bars = 10mm.


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Figure 7. Effect of the disruption of the HMG1 gene from Arabidopsis on the morphology of the ER. Arabidopsis plants of theSALK_061790 line heterozygous for the disruptedHMG1 gene were transformedwith GFP-HDEL. At the segregating generation T2, thedifferentiation zone of roots from 7- to 10-d-old plants was examined by confocal laser microscopy. Images were captured after flu-orescent excitation of GFP or propidium iodide (A, D, G, and I–L) under bright field (C and F), or with the merge of both channels (B andE). A to C, Morphology of the ER in the root of the heterozygous plant. The GFP-HDEL label is detected in the cortical ER, the nuclearenvelope, and more intensively, ER bodies. The nuclei (indicated by arrows) appear as fusiform structures. D to F, Morphology of the ER





in the process. In agreement with the dispensability of thecatalytic domain of HMGR, the induced OSER structuresdid not accumulate sterols. Our finding that sterols arebarely detected in OSER structures is consistent with theirexpected low level in ER membranes (Korn, 1969) andindicates that sterol accumulation is not required forOSER biogenesis, which is in contrast to a previousproposal (Lingwood et al., 2009). Our results indicate amorphogenetic rather than a catalytic role for HMGR inOSER biogenesis, but the proliferated membranes couldsubsequently accommodate active enzyme machineries.It was previously proposed that the synthesis of differentclasses of isoprenoids could be mediated by enzymaticarrays or metabolons containing specific HMGR iso-zymes in particular ER domains (Chappell, 1995). Plantand animal cells engaged in high production of definiteisoprenoid products typically contain OSER structures(Schnepf, 1974; Fawcett, 1981) that may contain the pre-dicted metabolons.

We found that the three-Arg motif conserved in theN-terminal region of plant HMGR is an absolute re-quirement to induce ER proliferation and morpho-genesis. In animal cells, Arg residues located betweenpositions 2 and 5 of type II membrane proteins constitutean ER retention signal (Schutze et al., 1994), and a motifof Arg residues close to the N terminus was shown toaccomplish an ER retention role as well in plant cells(Boulaflous et al., 2009). In contrast, the Arg tag of plantHMGR is not required for retention in the ER but rep-resents an ER morphogenic signal. Nevertheless, thethree basic residues are only a part of the N-terminalmotif conserved in plant HMGR, and the two hydro-phobic segments and the short lumenal region of themembrane domain are also highly conserved (Camposand Boronat, 1995). These elements may also contributeto HMGR-driven ER proliferation and morphogenesis.The requirement of the three-Arg sequence for ER mor-phogenesis indicates that the same motif identified in 194of 305 analyzed plant HMGRs (64% of the total) shouldaccomplish an equivalent role. We have experimentallyconfirmed Arabidopsis HMGR1S and HMGR2 asmembers of this group. Other HMGR variants carryingtwo Arg residues of three Arg positions or with con-servative changes in the basic motif (an additional 26%)will likely perform the same or a very similar function.However, those HMGRs with no N-terminal Arg motif

(the remaining 10%) represented by Arabidopsis HMGR1Lmay be unable to induce ER proliferation and OSERbiogenesis. Thus, ER morphology may be regulated byexpression of different HMGR isoforms in plant cells.The fact that OSER biogenesis is driven by plant HMGRthrough conserved sequences, its occurrence in all testedplants and cell types, its reversibility, and its compati-bility with cell function and plant development reinforcethat ER morphogenesis is naturally controlled byHMGR. This was confirmed experimentally in Arabi-dopsis, where the disruption of the HMG1 gene severelyaffected the morphology of the nuclear ER.

In the three eukaryotic kingdoms, HMGR exerts keyregulation in sterol biosynthesis through its catalytic do-main and triggers ER proliferation and OSER biogenesisthrough its membrane domain. This coincidence has beenpreserved along evolution, underlining its importance foreukaryotic life. Remarkably, the membrane domain ofHMGR has retained its morphogenic capacity, despitelow sequence conservation. OSER biogenesis driven byHMGR was shown here in plants and observed previ-ously in mammalian cells and yeast in three cases usinghomologous expression systems (Jingami et al., 1987;Wright et al., 1988). In addition, OSER structures wereinduced by expression of yeast HMGR in mammaliancells or mammalian HMGR in yeast (Wright et al., 1990),indicating common mechanisms in this morphogenicphenomenon. Also conserved is the subcellular locationfor ER proliferation. OSER biogenesis driven by HMGRhas been shown to initiate at the nuclear envelope inmammalian cells (Pathak et al., 1986), and HMGR-induced lamellae (thus named karmellae) also appeararound the nucleus in yeast (Wright et al., 1988). In plantcells, OSER structures emerge from ER network junctionsand around the nucleus, where they mostly accumulate.The morphogenic action of HMGR at the nuclear enve-lope was confirmed by disruption of the HMG1 genefrom Arabidopsis, which caused ER aggregation at thissite. The fact that plant HMGR requires a highly con-served motif to control morphogenesis of the ER suggeststhat other eukaryotic HMGRs may have alternative se-quences with equivalent function.

OSER structures induced by 1S:GFP or 1S:GFPm inplant cells are similar to those observed in animals.Higher eukaryote OSER structures are formed bytightly packed interconnected regions of crystalloid,

Figure 7. (Continued.)in the root of the homozygous SALK_061790 mutant plant. The GFP-HDEL label mainly distributes in ER bodies around the nucleus.The nuclei (seen from the front; arrows) have a circular appearance with an irregular outline. G, Nuclear region in cortex cells from theSALK_061790 hmg1 mutant. The image corresponds to a Z projection of 15 sections from the GFP channel taken every 0.85 mm. TheER bodies accumulate around the nucleus (yellow N). The individual sections are shown on bright field in Supplemental Movie S2. H,Phenotype of SALK_061790 homozygous hmg1mutant (left) and wild-type (right) plants 10 d after growth at 22˚C. The mutant plant hasa delayed development in both aerial and root organs and is still at the two cotyledon stage. I, Nuclear region in cortex cells from thewild type. The center of the nucleus is indicated with a yellow N. The image corresponds to a Z projection of 15 sections from the GFPchannel taken every 0.52 mm. The individual sections are shown on bright field in Supplemental Movie S3. J, DAPI staining and GFPdetection of homozygous mutant root. The image corresponds to a Z projection of seven sections. K and L, Propidium iodide stainingand GFP detection of homozygous mutant root. L corresponds to the XZ scanning section indicated by the dashed line in K. The nuclearER body aggregates localize in cortex cells just below the epidermal layer. A to F, K, and L correspond to single scanning sections. G, I,and J are Z projections. Bars = 20 mm (A–F, K, and L), 10 mm (G, I, and J), and 3 mm (H).


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lamellar, and whorled smooth ER membranes andmorphologically more complex than the lamellarOSER formations observed in yeast. Also, in a closesimilarity, animal and plant cell OSER structures con-tain membrane stacks with two distinct alternatingspaces: the electron-translucent lumen, which shows ageneral ordered pattern but can be distended anddisordered in some regions, and the more electron-dense narrow cytosolic area that is delimited by adja-cent cisternae, which has a very constant width. Theelectron-dense aspect and even spacing of the cytosolicarea indicate accumulation of regularly disposed ma-terial, likely proteins. It was suggested that the widthof the cytosolic space depends on the size of theoverexpressed OSER-inducing protein (Snapp et al.,2003), but this is not the case in OSER structures in-duced by plant HMGR. Because 1S:GFPm does notdimerize, other ER resident or cytosolic ER-interactingproteins should be involved in the close associationof membranes. Interestingly, freeze-fracture EM oftransfected human cells uncovered a high density ofintramembrane particles in the cytosolic face of theOSER membranes and a much lower number of sim-ilar structures in the lumenal face (Anderson et al.,1983). It is tempting to speculate that the observedprotoplasmic particles may correspond to proteincomplexes mediating membrane apposition.It was previously noticed that the formation of OSER

structures in mammalian cells correlated with the ex-pression level of the OSER-inducing protein. When cy-tochrome b5 was highly expressed, it induced OSERstructures, but at lower expression, the ER network wasnot disturbed (Snapp et al., 2003). Thus, it was arguedthat OSER biogenesis merely results from overexpressionof ERmembrane proteins (Yamamoto et al., 1996; Li et al.,2003; Snapp et al., 2003). Certainly, OSER biogenesis in-duced by 1S:GFP is associated to a high accumulation ofthe chimera. However, we found that OSER structuresalso accumulate endogenous HMGR protein, with con-comitantly reduced HMGR transcript levels. Thus, themembrane availability induced by 1S:GFP somehowstabilizes the chimera and also, the endogenous HMGR,which was previously found in several plants to be sub-mitted to ongoing degrading processes (Korth et al., 2000;Leivar et al., 2011; Pollier et al., 2013). Other endogenousand heterologous ER membrane proteins are also stabi-lized in OSER structures (Kochevar and Anderson, 1987;McLaughlin et al., 1999). Chimeras 1L:GFP, DDLRRR1S:GFP, andDRRR1S:GFP are nearly identical to 1S:GFP, withthe only difference of the naturally extended N-terminalregion or the elimination of the conserved N-terminalmotif. In agreement with the stabilization of HMGR inOSER structures, the observed lower expression levels of1L:GFP, DDLRRR1S:GFP, and DRRR1S:GFP might notbe the cause but a consequence of their inability to induceER proliferation and morphogenesis.Our data indicate that ER proliferation and OSER bio-

genesis can be induced by ER resident proteins, such asHMGR, carrying conserved short-peptide motifs thatfunction as ER morphogenetic signals. Previous work

showed that overexpression of oligomerizing ER pro-teins leads to conversion of preexisting ER into OSERstructures without significant membrane proliferation(Snapp et al., 2003). On this basis, we propose a two-stepmodel for OSER biogenesis in eukaryotic cells: (1) ERproliferation induced by definite sequences and struc-tural elements of particular ER resident proteins, such asHMGR, and (2) membrane stacking promoted by olig-omerization of ER resident proteins. This model wouldallow induction of OSER structures by overexpression ofoligomerizing ER proteins, skipping the first of twoproposed steps. Abundant ER resident proteins (notnecessarily the ones inducing ER proliferation) couldmediate the stacking of membranes. In support ofthis, OSER biogenesis was triggered by expression of1S:GFPm and 1S constructs, which do not have a cyto-solic oligomerizing domain. The 1S-GFPm OSER struc-tures had tightly stacked ER membranes with narrowcytosolic space that are indistinguishable from those in-duced by 1S:GFP in plant cells (this work) or other oli-gomerizing proteins in animal cells (Snapp et al., 2003).The stabilization of ER resident proteins in OSER struc-tures detected here for endogenous HMGR could be fa-vored by the availability of membrane space or theisolation from external proteases. The stabilized proteinswould then contribute to OSER biogenesis by promotingmembrane association. The membrane stacking stepmight be energy dependent, because ATP deprivationseverely disrupted the ordered pattern of proliferated ERmembranes (Lingwood et al., 2009). Sustained expres-sion of the OSER-inducing protein may be needed tomaintain OSER structures, because we found that dis-appearance of 1S:GFP in plant cells allows reversion tonormal ER network. Thus, our observations suggest thatOSER formation and disassembly may be controlled in asimple way by determining the expression level ofmorphogenic HMGR isoforms.

MATERIALS AND METHODS

Plant Material

Arabidopsis (Arabidopsis thaliana) seeds were surface sterilized by washingthree times with 70% (v/v) ethanol and three times with 100% (v/v) ethanol,sowed in petri dishes with Murashige and Skoog medium (Murashige andSkoog, 1962), and vernalized at 4°C for 3 d. Seedlings were grown under long-day conditions (16-h-light/8-h-dark cycle) at 22°C to 24°C. Arabidopsis plantswere grown on a perlite:vermiculite:sphagnum (1:1:11) mixture in the samelight regime. The undifferentiated T87 cell line from Arabidopsis was culturedas indicated (Axelos et al., 1992). The SALK_061790 mutant from Arabidopsis(Myouga et al., 2010) carrying a T-DNA insertion in the first exon of geneHMG1 (AT1G76490) was obtained from the European Arabidopsis StockCentre. Nicotiana benthamiana was grown on perlite-vermiculite-sphagnum ina greenhouse under natural day conditions. Fresh onions (Allium cepa) andleeks (Allium porrum) were purchased in the market and used a few hours laterfor biolistic transfection.

Constructs and Plant Transfection

For biolistic transfection, constructs were cloned in plasmid pGFPau (Leivaret al., 2005). For agroinfection or generation of Arabidopsis transgenic plants,constructs were cloned in plasmid pPCV002 (Ferrando et al., 2001) providedby Alejandro Ferrando (Instituto de Biología Molecular y Celular de Plantas,





Valencia, Spain). In both plasmids, transcription is under control of the cau-liflower mosaic virus 35S promoter, which confers high constitutive expres-sion. Constructs were prepared by conventional PCR and cloning techniquesas indicated in Supplemental Table S1 using the oligonucleotides listed inSupplemental Table S2. The pPCV002 derivatives were transformed intoAgrobacterium tumefaciens strain GV3101 pMP90RK (Koncz and Schell, 1986).Transgenic Arabidopsis plants were generated by the floral dip method(Clough and Bent, 1998) using the transformed A. tumefaciens cells. Transientexpression in Arabidopsis leaves, onion scale leaves, and leek scale leaves wasachieved by biolistic transfection with pGFPau derivatives. Transient expres-sion in the Arabidopsis T87 cell line (Ferrando et al., 2001) and N. benthamianaleaves (Van der Hoorn et al., 2000) was achieved by infection with A. tumefaciens(agroinfection) carrying the appropriate pPCV002 derivatives. Biolistic transfec-tion was performed with a Biolistic PDS-1000/He System (BioRad) with a900-pounds-force per square inch rupture disc (BioRad) as described (Lois et al.,2000). Samples were observed 18 to 48 h after biolistic transfection and 72 to 96 hafter agroinfection.

Determination of Transcript and Protein Levels

HMG1 and HMG2 transcripts were quantified by quantitative reversetranscription and real-time PCR as described (Leivar et al., 2011) using theAt4g26410 transcript as a normalization reference. The primer sets and theamplicon sizes for the transcripts were as follows: At4g26410, primers 264Fand 264R (81 bp); HMG1, primers 764F and 764R (85 bp); and HMG2, primersHMG2F and HMG2R (92 bp). Primers are listed in Supplemental Table S2.HMGR protein levels were determined by immunoblot with anti-CD1-i anti-body as described (Leivar et al., 2011).

Determination of ER Morphology in theSALK_061790 Mutant

To determine the morphology of the ER, the SALK_061790 line from Arabi-dopsis carrying a T-DNA insertion in the HMG1 gene was transformed with theGFP-HDEL marker of the ER lumen. Because the homozygous disruption of theArabidopsis HMG1 gene severely affects development and fertility, transforma-tion was performed on heterozygous plants. A. tumefaciens strain GV3101 pMP90pVK-Her6 encoding GFP-HDEL (Batoko et al., 2000) was provided by Ian Moore(University of Oxford, UK). Transformants were selected in generation T1 byresistance to hygromycin and the appearance of GFP fluorescence in the wholeplant. In general, the fluorescence was more intense in the root. Generation T1was self-crossed to obtain T2 seeds, which were germinated in one-half-strengthMurashige and Skoog medium. After 7 d of growth, homozygous mutants werestill at the two-cotyledon stage, whereas homozygous wild-type and heterozy-gous plants had developed true leaves. The root was sectioned between days 7and 10 for immediate observation with a confocal microscope, and the rest of theplant was transferred to a fresh plate for additional growth before confirmation ofthe genotype by PCR. The wild-type allele was identified with primers H1.2F andH1.3R (547-bp amplification product), and the mutant allele was identified withprimers LB6 and H1.3R (358-bp product).

Stereomicroscopy and Confocal Microscopy

Arabidopsis seedlings were observed with the stereomicroscope OlympusSZX16 (with excitation at 460 to 495 nmand emission above 510 nm) todetectGFPand chlorophyll. Arabidopsis seedlings were stained with filipin III (Boutté et al.,2011), DAPI (Kapuscinski, 1995), or propidium iodide (González-García et al.,2011) and observed with the confocal microscope as reported. Confocal lasermicroscopy was performed with spectral microscope Olympus FV1000 (objec-tives UPLSAPO 603 O, numerical aperture: 1.35 and UPLSAPO 603 W, nu-merical aperture: 1.20) or Leica TCS SP5 (objectives HCX PL APO CS 40.03 1.25oil UV, HCX PL APO 63.03 1.20W corrected for UV, and HCX PL APO CS 63.031.20 water UV) at room temperature. Fluorophores were detected with thefollowing excitation and emission wavelengths: GFP (excitation = 488 nm,emission = 500–545 nm), T3RE (excitation = 543 nm, emission = 555–700nm), Filipin-sterol complexes (excitation = 364 nm, emission = 400–484 nm),DAPI (excitation = 405 nm, emission = 425–490 nm), propidium iodide(excitation = 559 nm, emission = 570–670 nm), Alexa Fluor-488 (excitation =488 nm, emission = 500–545 nm), and Alexa Fluor-594 (excitation = 559 nm,emission = 570–670 nm). Images were acquired with the software FV10-ASW 4.1 (Olympus) and LAS AF 2.7.3.9723 (Leica) and processed withthe software ImageJ 1.48v (http://imagej.nih.gov/ij).

Antibodies

HMGR was detected with immunosubtracted rabbit serum recognizing itscatalytic domain (anti-CD1-i; Leivar et al., 2011) as the primary antibody at1:1,000 for immunoblot, 1:500 for whole mount, and 1:1,000 for transmissionEM. Anti-rabbit IgG secondary antibodies for HMGR detection were codeNA934V (GE Healthcare) coupled to horseradish peroxidase at 1:5,000 forimmunoblot, code Ab150068 (Abcam) coupled to Alexa Fluor-594 at 1:1,000for whole mount, and code 111-205-144 (Jackson Immunoresearch) coupled toa 12-nm gold particle at 1:30 for transmission EM. GFP was detected withAb-5450 (Abcam) as the primary antibody at 1:1,000 for whole mount andtransmission EM. Anti-goat IgG secondary antibodies for GFP detection werecode Ab150133 (Abcam) coupled to Alexa Fluor-488 at 1:1,000 for wholemount and code 705-215-147 (Jackson Immunoresearch) coupled with an 18-nmgold particle at 1:15 for transmission EM.

Immunolocalization in Whole Mount

Whole-mount in situ immunolocalization was done as described (Saueret al., 2006) with modifications. After fixation in 4% (w/v) paraformaldehyde,seedlings were incubated in methanol to remove chlorophylls. Five cycles offreezing and thawing of the seedlings on glass slides were performed topermeate tissue followed by incubation with 2% (w/v) Driselase (D8037;Sigma) to allow for penetration of antibodies through the plant cell wall. Afterblocking with 3% (w/v) bovine serum albumin (BSA) solution, incubationwith one or two primary antibodies simultaneously (anti-CD1-i at 1:500 and/or Ab-5450 at 1:1,000) was performed directly on the slides. Samples were thenwashed with phosphate-buffered saline (PBS) and incubated with one or twosecondary antibodies. Secondary antibody for HMGR detection (Abcam codeAb150068; Alexa Fluor-594) was used at 1:1,000. Secondary antibody for GFPdetection (Abcam code Ab150133; Alexa Fluor-488) was used at 1:1,000. Afterwashing with PBS, samples were sealed for observation at the confocalmicroscope.

Transmission EM Ultrastructural Studies

Explants from N. benthamiana leaves or Arabidopsis seedlings were re-moved under a stereomicroscope and transferred to glass vials filled with1.5% (v/v) paraformaldehyde and 1.5% (v/v) glutaraldehyde in phosphatebuffer (pH 7.4) containing 2 mM CaCl2. The vials were incubated at 4°C for48 h. After washing with the phosphate-CaCl2 buffer, samples were postfixed for3 h at 4°C with 1% (w/v) osmium tetroxide and 0.8% (w/v) potassium fer-ricyanide in the same buffer. Samples were subsequently dehydrated in ace-tone, infiltrated with Spurr resin for 4 d, embedded in the same resin, andpolymerized at 60°C for 48 h. Cell integrity was confirmed at the light mi-croscope. Ultrathin sections were obtained using an Ultracut UC6 Ultrami-crotome (Leica) and mounted on Formvar-coated copper grids. Samples werestained with 2% (w/v) uranyl acetate in water and lead citrate. Samples wereobserved in a JEM-1010 Electron Microscope (JEOL) equipped with a CCDCamera SIS Megaview III and the AnalySIS software.

Transmission EM Immunocytochemical Studies

Explants from Arabidopsis seedlings were cryoimmobilized by high-pressure freezing using an EM Pact (Leica) with yeast (Saccharomycescerevisiae) paste as the filler. Freeze substitution of frozen samples was performedin an Automatic Freeze Substitution System EM AFS (Leica) with methanolcontaining 0.5% (w/v) uranyl acetate at 290°C for 3 d. On the fourth day, thetemperature was increased slowly by 5°C per hour to 250°C. At this tempera-ture, samples were rinsed in acetone, infiltrated, and flat embedded in LowicrylHM20 for 10 d. Ultrathin sections were picked up on Formvar-coated nickelgrids. Sample-containing grids were incubated on drops of PBS with 5% (w/v)BSA for 20 min at room temperature. After removal of the washing solution,drops of PBS with the polyclonal primary antibody (anti-CD1-i or Ab-5450 at1:1,000) and 1% (w/v) BSA were added and incubated for 2 h. Grids werewashed three times for 30 min with a drop of PBS with 0.25% (v/v) Tween 20 andincubated for 1 h in drops of PBS with the secondary antibody and 1% (w/v)BSA. Secondary antibody for HMGR detection (code 111-205-144; JacksonImmunoresearch; 12-nm gold particle) was used at 1:30. Secondary antibody forGFP detection (code 705-215-147; Jackson Immunoresearch; 18-nm gold particle)was used at 1:15. Grids were washed three times with a drop of PBS for 5 minand two times with distilled water and air dried. In control assays for nonspecific


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http://imagej.nih.gov/ij


binding of the gold-conjugated antibody, the primary antibody was omitted.Sections were stained with 2% (w/v) uranyl acetate in water and lead citrate andobserved in a JEM-1010 Electron Microscope (JEOL) with an SIS Mega View IIICCD Camera.

Bioinformatics

The N-terminal motif of plant HMGR was obtained with MEME softwareversion 4.9.1 (Bailey et al., 2009), which is accessible at http://meme-suite.org/ using the defect parameters.

Sequence data from this article can be found in the Arabidopsis GenomeInitiative and GenBank databases under accession numbers HMGR1L(At1g76490 and AAR83122), HMGR1S (At1g76490 and AAF16652), andHMGR2 (At2g17370 and AAA67317).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. ER morphology after expression of 1S:GFP and 2:GFP in Arabidopsis cells.

Supplemental Figure S2. ER morphology after expression of 1S:GFP, 2:GFP, and 1L:GFP in Arabidopsis cell culture.

Supplemental Figure S3. ER morphology after expression of 1S:GFP, 2:GFP, and 1L:GFP in onion or leek epidermal cells.

Supplemental Figure S4. The 1S:GFP-containing aggregates belong to the ER.

Supplemental Figure S5. Controls of immunodetection and organellemorphology.

Supplemental Table S1. Preparation of DNA constructs.

Supplemental Table S2. Oligonucleotides.

Supplemental Data Set S1. N-terminal sequence of plant HMGR.

Supplemental Movie S1. Physical connection between the ER network andER-derived aggregates.

Supplemental Movie S2. Morphology of the ER around the nucleus in theArabidopsis hmg1 mutant.

Supplemental Movie S3. Morphology of the ER around the nucleus inwild-type Arabidopsis.

ACKNOWLEDGMENTS

We thank the European Arabidopsis Stock Centre for seed stocks; BenjaminGlick, Patrick Gallois, Alejandro Ferrando, and Ian Moore for the plasmid gift;Montse Amenós for help in image processing; Albert Boronat for critical discus-sion; and the staffs of the Confocal Microscopy Service of Centre de Recerca enAgrigenòmica, the Advanced Optic Microscopy Unit of the Centres Científics iTecnològics de la Universitat de Barcelona (Faculty of Biology), the ElectronMicroscopy Units of the Centres Científics i Tecnològics de la Universitat deBarcelona (Campus Casanova and Campus Pedralbes), the Serveis de CampsExperimentals of Univertat de Barcelona, and the Plant Growth Facility of Centrede Recerca en Agrigenòmica for support in microscopy and plant culture.

Received April 22, 2015; accepted May 23, 2015; published May 26, 2015.

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