https://doi.org/10.3390/d15020170
“The lichenized green microalga Trebouxia lynnae Barreno has been recently described and is considered a model organism for studying lichen chlorobionts. Its cellular ultrastructure has already been studied in detail by light, electron, and confocal microscopy, and its nuclear, chloroplast and mitochondrial genomes have been sequenced and annotated. Here, we investigated in detail the ultrastructure of in vitro grown cultures of T. lynnae observed by Low Temperature Scanning Electron Microscopy (LTSEM) applying a protocol with minimum intervention over the biological samples. This methodology allowed for the discovery of ultrastructural features previously unseen in Trebouxiophyceae microalgae. In addition, original Transmission Electron Microscopy (TEM) images of T. lynnae were reinterpreted based on the new information provided by LTSEM. The nucleolar vacuole, dictyosomes, and endoplasmic reticulum were investigated and reported for the first time in T. lynnae and most likely in other Trebouxia lineages.”
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2.2. Low Temperature Scanning Electron Microscopy (LTSEM)
For LTSEM, microalgal colonies plus the underlying agar medium or acetate discs were cut into squares of around 2 × 2 mm from the centre of the colony and attached to a cryo-holder using colloidal graphite. Samples were plunge frozen in LN2 slush and transferred with a transfer rod module into the cryo-preparation system (PP3010T, Quorum Technologies, Sussex, UK). Samples were mechanically freeze-fractured and then freeze-etched by sublimation for 15 to 25 min at −90 °C. The time of sublimation was adjusted to the estimated water content of the sample and the type of structures aimed at imaging. A thin layer of platinum was sputtered onto the specimens for 10 s and afterward transferred into a Field Emission Scanning Electron Microscope (FESEM ZEISS Ultra-55, Carl ZEISS SMT, Oberkochen, Germany). Images were recorded at an accelerating voltage of 1.5 kV. The images are photographic negatives; hence, protuberant elements of the fractured/etched surface are most heavily coated with platinum and appear white.
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Within the
T. lynnae colony, different types of cells were recognized both by LTSEM and by TEM. Young cells (6 ± 2 μm; A,B) presented shallowly lobed chloroplasts displaying a low number of lobes and a single pyrenoid. Mature vegetative cells (10 ± 1 μm; C,D) had shallowly lobed chloroplasts and a single impressa-type pyrenoid [
9]. In both young and mature vegetative cells, a high number of vesicles was observed, and a moderate number of mitochondria could be distinguished, although they were inconspicuous. Mature vegetative cells displayed a big nucleus with a clearly distinguishable nucleolus.
Figure 2. LTSEM (A,C) and TEM (B,D) of Trebouxia lynnae vegetative cells. (A,B) Young vegetative cells. (C,D) Mature vegetative cell. Chloroplast (c), pyrenoid (p), vesicles (v), starch grains (s), mitochondria (m), and nucleus (n) are indicated with letters. Scale bars: (A–D) 1 µm.
Two types of sporangia were observed ( and ). One in which spores with cell walls are symmetrically arranged (11–13 μm; ), presumably autosporangia (i.e., mitotic sporangia carrying cell-walled spores that resemble the morphology of vegetative cells). The chloroplast of the autospores was of the shallowly lobed type [
9], with a low number of lobes (A,B). The pyrenoid was recognizable as impressa-type (A). Before opening, the spaces between cells are filled with an aqueous substance (A,B) that is missing when the envelope is opened (C). The autosporangia envelope was frequently persistent, even at the maturity of the daughter cells (D,E).
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