Analysis version 3

A 1 cm square of dorsal skin was excised from the mouse, placed flat on a piece of paper towel and fixed in 4% paraformaldehyde and 2% glutaraldehyde in 0.05M cacodylate buffer (pH 7.4) overnight at room temperature. The tissues were then rinsed four times for 10 min each in cacodylate buffer and post-fixed and stained with 1% osmium tetroxide and 1.5% potassium ferricyanide in 0.1 M cacodylate buffer for 1 h. Tissues were then washed four times in cacodylate buffer followed by dehydration once for 10 min each in ethanol at 30%, 50%, 70%, 80%, 90% and 95% followed by three times for 20 min each in 100% anhydrous ethanol, and twice for 10 min each in propylene oxide. Following dehydration, the tissues were infiltrated with increasing concentrations of Agar 100 resin (20%, 50%, 75% and 100%) in propylene oxide for 16 h per step. The tissues were then embedded in fresh resin and allowed to polymerize in a 60°C oven for 48 h. The embedded tissue blocks were sectioned with a diamond knife on a Leica Reichert Ultracut S microtome, and ultrathin sections (80 nm) were collected onto 200 mesh, carbon/formvar-coated copper grids. The sections were then sequentially stained with uranyl acetate and lead citrate for 10 min each and viewed with a Tecnai 12 100 kV TEM (Phillips) equipped with a MegaView II CCD camera and Analysis® version 3.0 software (SoftImaging System).

Vesicles were isolated from mitochondria and resuspended in SH buffer (0.6 M sorbitol, 20 mM hepes pH 7.4). The vesicles were adsorbed to Carbon–Formvar‐coated copper grids. Grids were stained with 1% (w/v) uranyl acetate and air‐dried. Samples were viewed with Tecnai 12 TEM 100 kV (Phillips, Eindhoven, the Netherlands) equipped with MegaView II CCD camera and Analysis^® version 3.0 software (Soft Imaging System GmbH, Münstar, Germany).

For the analysis of plant morphology, plant tissues (i.e., leaves and roots) where obtained from 5-day-old Arabidopsis plants grown on MS-plates in the presence or absence of 10 μM m-tyrosine. The morphologies of mitochondria and plastids were established by transmission electron microscopy (TEM) of ultrathin plant sections, using Tecnai 12 TEM 100 kV (Phillips, Eindhoven, the Netherlands) microscope equipped with MegaView II CCD camera and Analysis^® version 3.0 software (SoftImaging System GmbH, Münstar, Germany), at the Bio-Imaging unit of the Institute of Life Sciences (The Hebrew University of Jerusalem). The relative densities (i.e., pixel intensities) of thylakoid grana membrane stacks and the average surface area of mitochondria have been manually evaluated from TEM images of ultrathin sections of 5-day-old plantlets grown in the absence or presence of m-tyrosine, using the ImageJ software (Version 1.52a) (Jensen, 2013 (link)). Student's t-test was performed to determine significant differences (P ≤ 0.05).