Tecnai g2 sphera

An electron microscopic analysis was performed using a Tecnai Sphera G2 (FEI Company), as previously described [18 (link)]. Briefly, the plant material was fixed by immersion in a 50 mM cacodylate buffer (pH 7.2) for 6 h at RT, then it was washed in a cacodylate buffer and twice in distilled water. The cacodylate buffer contained 0.5% (v/v) glutaraldehyde and 2.0% (v/v) formaldehyde. Next, the samples were fixed in 1.0% (v/v) osmium tetroxide for 1 h at RT, washed twice in distilled water, dehydrated by passage through an acetone series (20–100%) and infiltrated with Spurr resin (Sigma Aldrich) initially 33%, then 66% and finally 100%.
The analysis was performed in three independent biological repetitions with at least 100 cells per repetition. The types of plastids were recognised according to the common description as initial undifferentiated proplastids, differentiating proplastids with few internal membranes and dense matrix, amyloplasts, etioplasts and chloroplasts.

Transmission electron microscopy images were obtained with a FEI Tecnai Sphera G2 (FEI, Hillsboro, Oregon, USA) microscope. For comparative histological and ultrastructural analysis, microwave proceeded fixation, substitution and resin embedding of rosette leaves was performed as specified in Table 1. Sectioning and microscopy analysis was carried out as described previously (Daghma et al., 2011 (link)).

High pressure freezing (HPF) with a Leica EMHPF (Leica Microsystems, Germany) for cryofixation of 15–17 DAP embryos was used to minimize structural alterations during sample preparation. Isolated embryos were loaded into aluminium platelets with a cavity of 0.2 mm and a paste of 1:1 (v/v) mixture of yeast (Arxula adeninivorans) and cyanobacteria (Synechocystis 6308) as filler. After covering with the flat surface of a 0.30 mm platelet, samples were frozen under a pressure of ∼2000 bar and transferred into an automated freeze substitution (FS) unit (Leica Microsystems, Bensheim, Germany). FS and resin embedding were carried out as described in Supplementary Information (Supplementary Table 8). Ultrathin sections (70 nm) were cut with a Leica Ultracut microtome. Prior to ultrastructure analysis in a Tecnai Sphera G² (FEI company, Eindhoven, Netherlands) at 120 kV, sections were contrasted in a LEICA EM STAIN with uranyl acetate for 30 min followed by an incubation in Reynolds’ lead citrate for 1.5 min.