Azur 2

EMs and NECs samples of all three investigated genotypes were collected after 10 days of multiplication for morphological and histo-cytological characterizations. Their morphology was documented using a SMZ 1500 stereomicroscope (Nikon, Tokyo, Japan). Fresh material was stained with either 0.4% (w/v) Trypan Blue (Sigma-Aldrich) as described by Vondráková et al. (2014) (link) for cell structure observations or Lugol (iodine-potassium iodide) solution for observation of cells’ starch contents using a Jenaval transmission light microscope (Zeiss, Jena, Germany). For histological studies, EMs and NECs samples were fixed, dehydrated and infiltrated with paraffin as described by Lelu-Walter et al. (2018) (link). Sections (12 μm thick) were stained with Alcian Blue and Nuclear Fast Red. Localization of phenolic compounds detected in vacuoles by Alcian Blue was also determined by staining with a 0.05% aqueous solution of Azur II and 1% Safranin in 50% ethanol (all dyes Sigma-Aldrich). Preparations were observed using a transmission light microscope. All images were captured using a DS-Fi3 camera (Nikon, Tokyo, Japan) and processed using NIS-Elements AR 5.0 software (Laboratory Imaging, Prague, Czechia).

For CFU assays, cells were seeded in limiting dilution, i.e., 2 cells/cm², incubated for 14 days, fixed with 4% formaldehyde, and stained with 0.1% Azur II (Sigma Aldrich, Taufkirchen, Germany) dissolved in Aq_dest for 20 min at room temperature (RT), air dried, and photographed with a SZH10 microscope (Olympus, Münster, Germany) equipped with a CCD Colour view III camera (Olympus, Münster, Germany). The resulting images were taken using the Cell Olympus cell Sens software version 1.5 (Olympus, Münster, Germany). Images were analyzed using the ImageJ plugin “analyze particles” following threshold adjustment. Stained colonies of ≥50 cell were scored as colony forming (CFU-F) and counted. CFU-F efficiency was calculated as follows: