Ultrastructural Analysis of Zinc Stress in Salvia sclarea Leaves

Pieces of S. sclarea leaves from plants exposed to 5 μM (control) or 900 μM (excess) Zn for 8 days were prepared for chemical fixation, as reported in [81 (link)]. Pieces were fixed firstly in a 3% paraformaldehyde + 3% glutaraldehyde solution buffered with 0.05 M sodium cacodylate at pH 7.0 at room temperature for 6 h, and subsequently, the leaf segments were post-fixed in 2% osmium tetroxide similarly buffered for 3 h. Afterwards, they were dehydrated in an acetone series, treated with propylene oxide and, finally, embedded in Durcupan ACM resin. An ultramicrotome (LKB 8801A, Stockholm, Sweden) equipped with a glass knife was used to obtained semi-thin sections (0.5–2 μm) that were stained with 0.5% (w/v) toluidine blue O and observed with a Zeiss Axioplan light microscope equipped with a digital AxioCam MRc 5 camera (Zeiss, Berlin, Germany). The stomatal density (No/mm²) was also evaluated in the leaf upper epidermis paradermal semithin sections [82 (link)] in both 5 μM and 900 μΜ Zn-exposed plants. Paradermal sections were obtained from 4 individual leaves from the central part of the leaf blade.