Zen 2014

For in vivo localization study of ANN1-GFP during plasmolysis, 3- to 4-day-old Arabidopsis plants expressing proANN1::ANN1:GFP or 35S::sGFP (control) constructs were used. Samples were mounted in half-strength MS medium (or in half-strength MS medium with FM4-64) in micro-chambers, and hypocotyl epidermal or primary root cells were documented at control conditions. Osmotic stress was induced either with 500 mM NaCl or 1 M mannitol solutions for hypocotyl, and 250 mM NaCl or 500 mM mannitol for root (in half-strength MS), which were applied by perfusion in a volume of about 100–150 µl. Imaging of ANN1-GFP or free GFP relocations during plasmolysis was done within the range of 1–40 min. Subsequently, deplasmolysis was realized by thorough sample perfusion with half-strength MS. Experiments were conducted by Airyscan CLSM 880 (Carl Zeiss, Germany) equipped with 20×/0.8 NA dry Plan-Apochromat objective (Carl Zeiss, Germany). Samples (hypocotyl in ANN1-GFP line) were imaged with a 488-nm excitation laser line and BP420-480 and BP495-550 emission filters for GFP detection. The image post-processing was done using ZEN 2014 software (Carl Zeiss, Germany). Additionally, Photoshop 6.0/CS, Microsoft Excel, and PowerPoint were used for final figure plates preparation.

The membrane styryl dye FM4-64 [N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl) pyridinium dibromide; Thermo Fisher Scientific, Waltham, MA, United States] was used for colocalization study of ANN1-GFP or free GFP (control) with the plasma membrane in root cells. Three-day-old Arabidopsis seedlings were placed in a drop of half-strength MS culture medium with 4 μM FM4-64 on a microscope slide in darkness for 15 min. After imaging under control conditions for approximately 15 min, the sample was washed by perfusion with the dye-free half-strength MS medium containing NaCl or mannitol and observed further. Specimens were observed by Airyscan CLSM 880 (Carl Zeiss, Germany) equipped with 20×/0.8 NA dry Plan-Apochromat objective (Carl Zeiss, Germany) and imaged using 488-nm excitation laser line and BP420-480 and BP495-550 emission filters for GFP detection, while FM4-64 was detected using beam splitter MBS 488/561 and BP420-480 and LP605. After the post-processing of images, fluorescence intensities of ANN1-GFP, free GFP, and FM4-64 were measured at the interface along two adjacent epidermal cells in the elongation zone of the primary root in ZEN 2014 software (Carl Zeiss, Germany). Photoshop 6.0/CS, Microsoft Excel, and PowerPoint were used for final figure plate preparation.

Five-day-old thalli expressing 2×Citrine driven by the MpSYP12B promoter were embedded in low-melt agarose gel and observed using a Light-sheet Z.1 microscope (Carl Zeiss) equipped with a water immersion lens (×5, numerical aperture = 0.16). The samples were excited at 488 nm (Argon 488 laser). Acquisition and construction of three-dimension images from multi-angle images were conducted using the ZEN2014 software (Carl Zeiss). The images were processed digitally with Imaris 8.2 (Bitplane) and Photoshop software.