Confocal microscopy and image analysis

For the detection of endogenous NO, 3-day-old seedlings were gravistimulated by 90° rotation and loaded with a 10 μM solution of the cell-permeable fluorescent probe 3-amino, 4-aminomethyl-2,7-difluorofluorescein diacetate (DAF-FM DA, Molecular Probes, USA) in 5 mM MES pH 5.7 containing 0.25 mM KCl and 1 mM CaCl₂ for 30 min. Before observation, seedlings were washed 3 times with the same buffer. Finally, DAF-FM DA-dependent fluorescence was detected in a confocal microscope (Nikon Eclipse C1 Plus) using a 20 x objective, NA = 0.75, an argon ion laser set at 488 nm; emission was collected between 500 and 530 nm. Six z-stacks were collected at 6 μm axial interval. Single pictures with no saturated pixels were combined into a sum of slices projection with the aid of FIJI bundle software (Schindelin et al., 2012). The fluorescence intensity of each individual root was quantified on digital images within independent regions of interests located in the lower and upper side of the epidermal root cells and in the outermost layer of the cortical root cells by using FIJI (Schindelin et al., 2012).

For the detection of PIN2-GFP, 3-day-old seedlings were gravistimulated by 90° rotation. Fluorescence was detected by confocal microscopy. Images were obtained with a Nikon Eclipse C1 Plus, 40 x objective, NA = 1.3 with an argon ion laser set at 488 nm; emission was collected between 500 and 530 nm. To obtain images under gravitropic stimulus we used a Zeiss LSM333 confocal microscope equipped with a vertical stage (InverterScope®) 20 x objective, NA = 0.8 with an argon ion laser set at 488 nm; emission was collected between 490 and 561 nm. Fluorescence intensity at the plasma membrane was quantified in the shootward and rootward sides of root cells from digital images with no saturated pixels using FIJI (Schindelin et al., 2012).