*4.9. Ultrastructural Analysis*

Embedded blocks for ultrastructural analysis were submitted to thin sectioning and cell integrity was confirmed at the light microscope. Ultrathin sections were obtained using an Ultracut UC6 Ultramicrotome (Leica) and mounted on Formvar-coated copper grids. Samples were stained with 2% ( *w*/*v*) uranyl acetate in water and lead citrate. Samples were observed in a JEM-1010 Electron Microscope (JEOL) equipped with a CCD Camera SIS Megaview III and the AnalySIS software. After capture, image brightness and contrast were adjusted with ImageJ 1.50i for a better visualization.

#### *4.10. Immunochemical Ultrastructural Analysis*

Explants from *Arabidopsis* seedlings were cryoimmobilized by high-pressure freezing using an EM Pact (Leica) with yeas<sup>t</sup> paste as the filler. Freeze substitution of frozen samples was performed in an automatic freeze substitution system (EM AFS (Leica)) with methanol containing 0.5% ( *w*/*v*) uranyl acetate at −90 ◦C for 3 d. On the fourth day, the temperature was raised by 5 ◦C per hour to −50 ◦C. At this temperature, samples were rinsed in acetone, infiltrated and flat embedded in Lowicryl HM20 for 4 d. Ultrathin sections were picked up on Formvar-coated nickel grids. 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 primary antibody (Ab-CD1-i or Ab-5450 at 1:1000) and 1% ( *w*/*v*) BSA were added and incubated for 2 h. Grids were washed three times for 30 min with a drop of PBS with 0.25% (*v*/*v*) Tween 20 and incubated for 1 h in drops of PBS with the secondary antibody and 1% ( *w*/*v*) BSA. Secondary antibodies (at 1:30) for HMGR detection were codes 111-205-144 (12-nm gold particle) or 111-215-144 (18-nm particle) from Jackson Immunoresearch (Cambridge, UK). The secondary antibody for GFP detection (code 705-215-147; 18-nm particle; Jackson Immunoresearch, Cambridge, UK) was used at 1:15. The grids were washed three times with a drop of PBS for 5 min and two times with distilled water and air-dried. In control assays for the nonspecific 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 and observed in a JEM-1010 electron microscope (JEOL) with an SIS Mega View III CCD camera.

#### **Supplementary Materials:** The following are available online at https://www.mdpi.com/1422-006 7/22/17/9132/s1.

**Author Contributions:** Conceptualization, N.C. and R.E.G.-T.; methodology, R.E.G.-T., C.L.-I. and N.C.; validation, R.E.G.-T. and N.C.; investigation, R.E.G.-T. and N.C.; resources, N.C.; data curation, N.C.; writing—original draft preparation, N.C.; writing—review and editing, N.C. and C.L.-I.; visualization, R.E.G.-T. and N.C.; supervision, J.C.F. and N.C.; project administration, N.C.; funding acquisition, J.C.F. and N.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Generalitat de Catalunya (grant 2017SGR710), Centres de Recerca de Catalunya (CERCA), the Ministerio de Asuntos Exteriores y de Cooperación-Agencia Española de Cooperación Internacional para el Desarrollo (predoctoral scholarship to R.E.G.-T.) and the Ministerio de Economía y Competitividad through the Severo Ochoa Program for Centers of Excellence in R&D 2016–2019 (SEV-2015-0533) and 2020–2023 (CEX2019-000902-S). The Article Processing Charge was financed by Ajuts de la Universitat de Barcelona per publicar en accés obert.

**Acknowledgments:** We thank Montse Amenós for help in image capturing and processing, Katharina Göttmann and Víctor Campos for the revision of English usage, the Confocal Microscopy Service of the Centre for Research in Agricultural Genomics (CRAG), the Advanced Optic Microscopy Unit of the Scientific and Technological Centers of the University of Barcelona (Faculty of Biology) and the Servei de Camps Experimentals de la Universitat de Barcelona and the Plant Growth Facility of CRAG.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
