**2. Results**

#### *2.1. Subcellular Location of Arabidopsis HMGR in WT and 1S:GFP Transgenic Plants*

Immunolocalization whole-mount studies in *Arabidopsis* cotyledon indicated that endogenous HMGR mostly localized inside HMGR vesicles ranging from 0.2 to 0.6 μm in diameter [18]. These studies were done with a crude rabbit polyclonal antibody raised against the catalytic domain of *Arabidopsis* HMGR1 (Ab-CD1), but it was later reported that this serum cross-reacts with *E. coli* proteins [48]. Before proceeding with a deeper localization analysis of HMGR, we wanted to confirm the whole-mount studies with an immunopurified fraction of the antibody (Ab-CD1-i) [48]. In this improved assay, we confirmed that *Arabidopsis* HMGR mostly localizes in vesicular structures of parenchymal cells, in close proximity with chloroplast (Figure 1a–c). Our observations sugges<sup>t</sup> that the HMGR vesicles can measure up to 2 μm in diameter, somewhat more than previously reported.

We subsequently performed immunochemical transmission EM studies of the HMGR vesicles, both in wild type (WT) and 1S:GFP-overexpressing *Arabidopsis* plants. We used the immunopurified serum Ab-CD1-i to detect endogenous HMGR, and a commercial antibody against GFP (Ab-5450) to detect the chimeric 1S:GFP. We found selective deposition of gold particles on the surface and the inside of the HMGR vesicles, denoting the presence of both endogenous HMGR and the 1S:GFP chimera (Figure 1d–f,l). The HMGR vesicles were associated in small groups connected by ER strands (Figure 1e,f,l). This connecting ER was also immunolabeled with the antibodies against HMGR and 1S:GFP (Figure 1e,l). The ultrastructural analysis uncovered that the HMGR vesicles were delimited by an ER membrane (Figure 1e). In addition, they possessed internal ER membranes (Figure 1d,f). Both the outer and internal membranes were recognized by the Ab-CD1-i (Figure 1d,e,l) and Ab-5450 (Figure 1f,l) antibodies. These results provide a rational explanation for the whole-mount detection of HMGR protein within HMGR vesicles.

As previously reported, overexpression of 1S:GFP in transgenic *Arabidopsis* plants induces ER proliferation and OSER structure biogenesis [19]. Whole-mount and immunochemical transmission EM analyses demonstrate colocalization of the 1S:GFP chimera and high levels of endogenous HMGR in the OSER formations (Figure 1g,l). Because of the presence of HMGR protein, we name them ER-HMGR domains. They have a disordered and heterogeneous crystalloid pattern, but with a characteristic layer of large loops in their external face and more compressed structures in the inside (Figure 1h,i,l). Precise deposition of immunogold particles indicates an abundance of 1S:GFP chimera and endogenous HMGR in the ER strands of ER-HMGR domains, both in the distended external loops and the internal membrane aggregates. High levels of the 1S:GFP chimera and endogenous HMGR were also detected in the ER network (Figure 1j,l) and nuclear envelope (Figure 1j,m). Few immunogold particles were observed inside the nucleus (Figure 1j), whereas no immunolabelling was found in the Golgi apparatus (Figure 1j), mitochondria (Figure 1h) or chloroplast (Figure 1d,h,j). In the negative control, no labelling was obtained without Ab-CD1-i and Ab-5450 primary antibodies (Figure 1k).

**Figure 1.** *Arabidopsis* HMGR localizes in the ER network, nuclear envelope, HMGR vesicles and ER-HMGR domains. (**<sup>a</sup>**–**<sup>c</sup>**) Whole-mount immunohistochemical analysis of cotyledon parenchymal cells from 6-day-old *Arabidopsis* WT seedlings (**a**) Immunodetection of HMGR with Ab-CD1-i and anti-rabbit IgG secondary antibody (Alexa Fluor 555, in red), visualized by confocal microscopy under dark (left) or bright fields (right). (**b**) Immunodetection of HMGR with Ab-CD1-i and antirabbit IgG secondary antibody (AlexaFluor 555, in red), and simultaneous detection of chlorophyll (in blue). (**c**) Negative control without the Ab-CD1-i antibody. The irregular corpuscles, 0.2 to 2 μm in length, correspond to HMGR vesicles. The elliptic bodies, 6 to 8 μm in diameter, correspond to chloroplasts. Images were obtained by Z-projection encompassing 3 (**a**), 10 (**b**) or 4 (**c**) μm in the Z-axis. Bars, 5 μm. (**d**–**f**) Immunochemical study of HMGR vesicles by transmission EM. Leaf samples from 10-day-old *Arabidopsis* WT or 1S:GFP seedlings were processed by HPF and embedded with Lowicryl

HM20. (**d**) HMGR was detected in cotyledon from WT seedlings with Ab-CD1-i and anti-rabbit-IgG (18 nm particle). (**e**) HMGR was detected in true leaf from 1S:GFP seedlings with Ab-CD1-i and anti-rabbit-IgG (12 nm particle). (**f**) Double immunolocalization of HMGR and 1S:GFP in true leaf from 1S:GFP seedlings. HMGR was detected with Ab-CD1-i and anti-rabbit-IgG (12 nm particle) and 1S:GFP was detected with Ab-5450 and anti-goat-IgG (18 nm particle). Black and blue arrowheads indicate, respectively, the external and internal membranes from HMGR vesicles. Red arrowheads indicate ER strands. Bars, 250 nm. (**g**) Whole-mount immunohistochemical analysis of cotyledon parenchymal cells from 6-day-old *Arabidopsis* 1S:GFP transgenic seedlings. 1S:GFP was detected with Ab-5450 and secondary antibody Alexa fluor 488 (green). HMGR was detected with Ab-CD1-i and secondary antibody Alexa fluor 594 (red). Images were obtained by Z-projection encompassing 12 μm in the Z-axis. Bar, 5 μm. (**h**–**<sup>m</sup>**) Immunolocalization of HMGR and 1S:GFP by transmission EM. True leaves from 10-day-old *Arabidopsis* WT seedlings were processed by HPF and embedded with Lowicryl HM20. (**h**–**j**) 1S:GFP was detected with Ab-5450 and anti-goat-IgG (18 nm particle). (**k**) Negative control without Ab-CD1-i and Ab-5450. (**l**) Double immunolocalization of 1S:GFP (18 nm particle) and HMGR (12 nm particle). (**m**) HMGR was detected with Ab-CD1-i and anti-rabbit-IgG (12 nm particle). Chloroplast (Chl). ER strands (red arrowheads). Golgi apparatus (G). Mitochondria (M). Nuclear envelope (blue arrowheads). Nucleus (N). Bars, 500 nm.

#### *2.2. Reversible Formation of ER-HMGR Domains*

To further characterize the biogenesis of ER-HMGR domains, we induced the transient expression of 1S:GFP in *Nicotiana benthamiana* leaves. The agroinfiltration approach allowed generalized and abundant expression of the 1S:GFP construct in leaf epidermis (Figure 2a). At day two after transfection, massive ER proliferation led to formation of OSER structures in the transfected tissue that were detectable even at low magnification (Figure 2a). Many small OSER structures appeared at ER network junctions and a single large OSER was formed around the nuclear envelope (Figure 2c,d). The 1S:GFPm construct, containing monomeric GFP, similarly induced small OSER structures at the network junctions and a large OSER aggregate around the nucleus (Figure 2b). As previously reported [19], this indicates that the membrane domain of *Arabidopsis* HMGR, and not its dimerizing GFP partner, induces ER proliferation and membrane association into OSER. In contrast to 1S:GFP, the 1S:GFPm chimera also accumulated in hypertrophied ER strands. Thick ER strands are usually present in epidermal cells and can be visualized with the ER-GFP luminal marker (Figure 2e), but become more prominent in the case of 1S:GFPm (Figure 2b). At day six after transfection, the expression of 1S:GFP was severely reduced. Concomitant with that, OSER structures virtually disappeared, with only some remnants around the nucleus and in the cytosol (Figure 2f). The resulting ER had the usual thick strands, although broad cisternae replaced the fine network (Figure 2f). ER cisternae are common in epidermal cells transfected with the luminal ER-GFP marker, although they have a smaller size (Figure 2e). Our observations in *Nicotiana* epidermal cells indicate that OSER biogenesis is reversible. The OSER structures are not a permanent consequence of transfection with 1S:GFP or 1S:GFPm, but can be replaced by quite normal ER when the levels of the chimeric protein decrease.

**Figure 2.** Reversible biogenesis of ER-HMGR domains in transfected *Nicotiana* cells. *Nicotiana* leaves were transfected with constructs encoding 1S:GFP (**<sup>a</sup>**,**c**,**d**,**f**), 1S:GFPm (**b**) or the control ER lumen marker ER:GFP (**e**) and epidermal cells were visualized by confocal laser microscopy. The expression time was 2 days (**a**), 3 days (**b**–**<sup>e</sup>**) or 6 days (**f**). The square regions indicated in (**b**,**d**,**f**) are shown enlarged on the right. Images are a single section (**a**) or Z-projections encompassing 10 (**b**), 21 (**c**), 7 (**d**), 12 (**e**) or 14 (**f**) μm in the Z-axis. Nucleus (N). OSER structures (white arrowheads). Thick ER strands (yellow arrowheads). Bars, 100 μm (**a**), 20 μm (**b**–**f**).

#### *2.3. ER-HMGR Domains Are Highly Dynamic*

To further inspect OSER structure morphology and dynamism, we obtained transgenic *Arabidopsis* plants stably expressing the 1S:GFP construct. A panoramic view of seedling root epidermis showed high expression of the 1S:GFP chimera accumulating at the ER (Figure 3a). The transgenic construct induced large OSER structures (up to 10 μm in diameter) around the nuclei and smaller OSER formations at ER network junctions (Figure 3a and Supplementary Movie 1). As previously reported [49,50], the ER network is highly dynamic with continuous strand movement and fusion or fission events. We found that

OSER structures are connected to the ER network and participate in its dynamism. Many strands of the ER network associate with OSER formations (Figure 3b). The ER strands rapidly connect to, slide along or separate from the OSER surface (Supplementary Movie 2). Small OSER structures migrate along fine or thick ER strands, whereas the large OSER formations have a more limited motion (Supplementary Movies 1 and 2). In the nuclear OSER, this movement may imply a brief separation from the nuclear envelope (Supplementary Movie 1). OSER structures have spherical-ovoid shapes with slight continuous variation (Supplementary Movies 2 and 3). The OSER borders are not sharp, but have a fluctuating blurry aspect (Figure 3b,c), suggesting the incorporation or emergence of GFP-labelled material (likely membranes) in the OSER surface (Supplementary Movies 2 and 3). We conclude that, in *Arabidopsis* cells, OSER structures are highly dynamic entities. They change in shape, have a moving surface and migrate intracellularly.

**Figure 3.** Characterization of ER-HMGR domains in *Arabidopsis* 1S:GFP transgenic plants. *Arabidopsis* 1S:GFP 9-day-old seedlings were analysed by confocal laser microscopy. The pictures show three channels (GFP, bright field and merge) of single sections from root epidermal cells that were subsequently characterized by live imaging: (**a**) panoramic view of root epidermis with large ER-HMGR domains indicated by white arrowheads (Supplementary Movie 1); (**b**) large ER-HMGR domain with dynamic connections to the ER network (Supplementary Movie 2); (**c**) nuclear ER-HMGR domains with changing spherical-ovoid shape (Supplementary Movie 3). Blurry fluctuating borders (yellow arrowheads). Nucleus (N). OSER structures (white arrowheads). Bars, 20 μm.

#### *2.4. The Fixation and Dehydration Method Severely Affects OSER Ultrastructure*

Our above confocal microscope observations of ER-HMGR domains do not fit the concept of OSER structures as rigid entities. Their tight, repetitive pattern, obtained after chemical fixation, contrasts with the flexibility and dynamism of OSER structures. To capture single states of OSER change at the ultrastructure level, we expressed 1S:GFP in *Nicotiana* leaf and submitted samples to high-pressure freezing (HPF) followed by freezesubstitution, to finally observe epidermal cells by transmission EM. We compared the HPF results with those of chemical fixation and subsequent dehydration at room temperature. With either method, OSER structures show a combination of crystalloid, lamellar and whorled membrane patterns (Figure 4a,b,g,h,j). In both cases, there is also a coincidence in cytosolic and luminal spaces. Luminal spaces correspond to the continuous internal cavity of the ER and have an electron-lucent aspect at transmission EM (Figure 4b,f,i,k). Cytosolic spaces result from the apposition of adjacent ER membranes and have a quite constant width, which is about 10–15 nm in both chemical and HPF fixation (Figure 4b,f,i,k). Cytosolic spaces have a darker aspect than luminal spaces, probably reflecting the presence of proteins that mediate the intermembrane attachment.

In spite of the above-mentioned coincidences, OSER structures have quite different overall morphology depending on the fixation method. The most remarkable feature of OSER formations after chemical fixation is the presence of highly repetitive convoluted membranes, which have a very different smooth and turgid aspect in HPF images (Figure 4a,c,e,h,i). The convoluted pattern is exclusive to chemically fixed crystalloid domains, whereas aligned membranes are present in whorled domains, both with HPF and chemical fixation (Figure 4b,j,k). However, the crystalloid (Figure 4a,c,d) and whorled (Figure 4b) domains are far looser after HPF than after chemical fixation (Figure 4h,j). The morphological heterogeneity generated by fixation is not due to the cytosolic spaces, which are always narrow and uniform, but occurs in the luminal spaces (Figure 4b,f,i,k). The small size and regularity of luminal spaces obtained after chemical fixation contributes to the repetitive pattern (Figure 4h,i). In contrast, HPF results in larger turgid luminal spaces, which are very variable in size and morphology (Figure 4e,f). After HPF and subsequent embedding by two alternative methods, the OSER morphology is disordered and heterogeneous, but has the above-mentioned row of large loops at the periphery and is internally more compact (see similarities of Figure 4a,c,d with Figure 1h,i,l).

To confirm the OSER ultrastructure resulting from chemical fixation, we expressed the membrane domain of *Arabidopsis* HMGR1S fused to monomeric GFP (chimera 1S:GFPm) in *Nicotiana* cells. Constructs 1S:GFP and 1S:GFPm differ in just one amino acid residue [19]. The 1S:GFPm chimera generated crystalloid, lamellar and whorled patterns with constant cytosolic distance between membranes and also regular luminal spaces (Figure 4l–o), similar to those obtained with 1S:GFP (Figure 4h–j). However, in OSER structures derived from 1S:GFPm the core of crystalloid domains was usually looser than the peripheral parts (Figure 4m). This was not observed in OSER structures derived from 1S:GFP, even when they were much larger (Figure 5e). We conclude that the dimerizing capacity of GFP may influence OSER membrane compaction during the chemical fixation process.

**Figure 4.** Ultrastructural analysis of ER-HMGR domains in *Nicotiana* epidermal cells. Three days after agroinfiltration with 1S:GFP (**<sup>a</sup>**–**k**) or 1S:GFPm (**l**–**<sup>o</sup>**), *Nicotiana* leaves were processed for transmission EM. (**<sup>a</sup>**–**f**) 1S:GFP-transfected leaves were submitted to HPF and embedded in Epon resin. (**a**) Panoramic view ER-HMGR domain with a crystalloid region in sagittal section and a whorled region in transversal section. (**b**) Magnified region of panel (**a**), to show the clear luminal spaces alternating with darker (denser for electrons) cytosolic spaces. (**<sup>c</sup>**,**d**) Panoramic view of crystalloid ER-HMGR domains with large loops surrounding the internal, more compact structure. (**e**) Magnified region of (**d**) with large loops. (**f**) Crystalloid structure with interspersed clear-luminal and dark-cytosolic spaces. The OSER cytosolic spaces are continuous with the cytoplasm (black arrows). Ribosomes are visible at the external face of the OSER structure. Occasionally, they are trapped in internal cytosolic spaces, near the periphery of the ER aggregate. (**g**–**k**) 1S:GFP-transfected leaves were submitted to chemical fixation and embedded in Spurr resin. (**g**) Panoramic view of crystalloid ER-HMGR domain. (**h**) Magnified region of (**g**) to show the repetitive smooth ER and a thin lamellar region on the left side. Ribosomes are excluded from the ER-HMGR domain. (**i**) Magnified regions of (**h**). (**j**) Whorled and crystalloid regions. (**k**) Whorled region with regular cytosolic and luminal spaces. (**l–o**) 1S:GFPm-transfected leaves were submitted to chemical fixation and embedded in Spurr resin. (**l**) Panoramic view of ER-HMGR domain with crystalloid, lamellar and whorled patterns. (**m**) Magnified region of (**l**) to show the looser region (circled) in the core of the ER-domain. (**n**) Magnified region of (**l**) to show the transition between whorled and crystalloid regions. (**o**) Magnified region of (**n**). Large membrane loops (black arrowheads). Crystalloid region (Cr). Cytosolic spaces (red arrowheads). Lamellar region (L). Luminal spaces (asterisks). Ribosomes (blue arrowheads). Whorled region (W). Bars, 1 μm (**<sup>a</sup>**,**c**,**d**,**g**,**h**,**l**–**<sup>n</sup>**), 200 nm (**b**,**e**,**f**,**i**–**k**,**<sup>o</sup>**).

**Figure 5.** Ultrastructural analysis of ER-HMGR domains in *Arabidopsis* parenchymal cells. Emerging true leaves from *Arabidopsis* 10-day-old seedlings transgenic for 1S:GFP were submitted to HPF or chemical fixation and embedded in Spurr resin. (**<sup>a</sup>**–**d**) Samples obtained by HPF. (**a**) Panoramic view of a crystalloid-lamellar ER-HMGR domain located between chloroplasts. (**b**) Magnified region of panel (**a**), to show the transition between crystalloid and lamellar regions. (**c**) Nuclear ER-HMGR domain with crystalloid and whorled patterns. (**d**) Magnified region of (**a**)**,** to show alternation of cytosolic (dark) and luminal (clear) spaces. Notice the narrow homogenous width of the cytosolic spaces and the broader and more variable size of the luminal spaces. (**<sup>e</sup>**–**i**) Samples obtained by chemical fixation. (**e**) Panoramic view of ER-HMGR domain with crystalloid, lamellar and whorled patterns. Notice the large size of the OSER structure (12 μm long). (**f**) Magnified region of panel (**e**), to show the transition between lamellar and crystalloid regions. (**g**) Detail of a crystalloid structure to show the repetitive pattern of dark cytosolic spaces and lighter luminal spaces. (**h**) Lamellar ER-HMGR domain. (**i**) Whorled ER-HMGR domain. Chloroplast (Chl). Crystalloid region (Cr). Cytosolic spaces (red arrowheads). Golgi apparatus (G). Lamellar region (L). Luminal spaces (asterisks). Mitochondria (M). Nucleus (N). Whorled region (W). Bars, 1 μm (**<sup>a</sup>**,**c**,**<sup>e</sup>**), 500 nm (**b**,**d**,**f**,**i**), 200 nm (**g**,**h**).

We also examined OSER ultrastructure in the emerging true leaves of the 1S:GFP *Arabidopsis* seedlings (10-day-old transgenic plants). We observed OSER structures with crystalloid, lamellar and whorled membrane patterns in parenchymal cells of both HPF and chemically fixed samples (Figure 5a,c,f). As mentioned above for *Nicotiana*, in *Arabidopsis* cells the three OSER ultrastructural patterns had a more relaxed morphology after HPF than after chemical fixation (Figure 5a,b,e,f). This is due to larger and more variable luminal spaces in HPF than in chemical fixation samples, whereas the dark cytosolic spaces had a similar width in both techniques (Figure 5b,d,g–i). The convoluted patterns observed in crystalloid domains after chemical fixation were absent in samples obtained by HPF (Figure 5d,g). Since HPF and chemical fixation were performed in parallel from the same samples, we conclude that the morphological differences of 1S:GFP-containing OSER were generated during the fixation process. Equivalent results were obtained in the two assayed systems, transfected *Nicotiana* leaves and *Arabidopsis* seedlings. As HPF immobilizes water and prevents its loss from the sample, the resulting transmission EM images are likely more similar to native OSER than those obtained by chemical fixation. In addition, the results obtained by HPF are more consistent with a dynamic view of the ER-HMGR domains observed with the confocal microscope.
