Application suite las software

Whole-mount in situ hybridization (WISH) experiments were performed as described [56 (link)] employing embryos and larvae previously fixed in 4% paraformaldehyde (PFA)/phosphate-buffered saline (PBS). Then, they were rinsed with a PBS-Tween solution, dehydrated in 100% methanol, and stored at −20 °C until processing. An 1166-bp Haspin fragment obtained by RT-PCR using has probe for 5′-AGTTGGAGCCTTGGATCTCC-3′ and has probe rev 5′-GGCAGTCCTCTCTTCCTGTT-3′ primers were cloned into the pGemT-Easy vector (Promega italia s.r.l., Milano, Italy). Sense and antisense RNA probes were transcribed using T7 and SP6 RNA polymerase (Roche), respectively, on templates linearized with either SalI or NcoI (New England Biolabs Inc, Ipswich, Massachusetts, USA). Riboprobes were labeled with digoxigenin using the “DIG-RNA Labelling Kit” (Roche). Images were acquired with a Leica DFC450C digital camera and the Leica Application Suite (LAS) software (Leica) on a Leitz DM RB microscope.

As a lipophilic styryl dye, FM 4-64 specifically stains the vacuolar membrane in yeast based on the method described by Journo D et al. in 2008 [45 (link)]. Yeast cells (control, L-UBB⁺¹, atg1Δ_control and atg1Δ_L-UBB⁺¹ strains) were cultured to mid exponential phase (OD_{600 nm} 0.5–0.6) in SD-His medium. 5 OD_{600 nm} units of cells were harvested and resuspended in 1 ml of YPD medium containing 4 μM of FM 4-64 dye (Invitrogen, USA). Cells were cultivated for 30 min at 30°C in the dark. Then cells were resuspended in 10 ml of YPD without FM 4-64 and incubated for 40 min at 30°C. After washing in 50 mM HEPES buffer (pH 7) twice, cells were resuspended in either SD-His medium or YNB (-N) medium containing 1 mM PMSF (Phenylmethylsulfonyl fluoride, Sigma Aldrich, USA) and 10 mM sodium citrate (pH 4.3). Rapamycin (MW 914.17, Cat no. R8781, Sigma Aldrich, USA) treatment was done in SD-His medium with a final concentration of 0.2 μM. After 4 h incubation at 30°C, cells were washed and resuspended in YNB (-N) medium containing 10 mM sodium citrate (pH 4.3) and visualized by Leica AF 6000 inverted fluorescence microscopy (Leica DMI4000B, Germany) using the DIC and FLUO-RFP filters. Images were processed with the Leica Application Suite (LAS) software and the numbers of cells with intravacuolar staining were quantified.

After staining, imaging of stomata was performed with a Nikon Eclipse 90i microscope equipped with a CCD camera using a TRITC filter Ex 540/25 DM 565 BA 605/55 (Nikon). Image analysis was performed using ImageJ software (https://imagej.nih.gov/ij/). For better visualization of the guard cells the option “sharpen” in ImageJ was used. The width and the length of the stomatal aperture were measured as shown on Fig 1A, and the stomatal aperture index (SAI) was calculated by division of the aperture width through the length. The SAI of at least 30 stomata per leaf was calculated, and three leaves per each treatment / time point were used for statistical analysis. Data were analyzed using Student’s t-test. The confocal images were captured under Leica SP8 microscope at following settings: excitation at 488 nm, emission 505–545 nm (green fluorescence); excitation at 561 nm, emission at 600–640 nm (red fluorescence), 20x objective, using Leica Application Suite (LAS) software.