Ti e platform

Stills of the actin and microtubule networks in epidermal pavement cells were taken by a Zeiss LSM880 microscope with a Plan-Apochromat 40 x/1.2 W objective and 488-nm argon laser for excitation, videos were taken by an inverted spinning disc confocal microscope (Yokogawa CSU-X1 on a Nikon Ti-E platform, laser box Agilent MLC400, camera Andor Ixon) with 100 x/1,45 O plan apochromatic objective, excitation laser line set at 488 nm, and image interval 1 s as described previously (Rosero et al., 2016 (link)). Cytoskeleton bundling and density were quantified as described previously (Higaki et al., 2010 (link)) with minor modifications (Rosero et al., 2013 (link); Rosero et al., 2019 (link)).
Cytoskeletal dynamics measurements were done in two biological replicates: with at least 40 cells from at least 20 videos analyzed using the QuACK method (Cvrčková and Oulehlová, 2017 (link)).
Actin networks in developing trichromes were visualized using an inverted spinning disc confocal microscope (Zeiss Axio Observer 7 microscope with a vertical stage equipped with a Yokogawa CSU-W1 spinning disk unit and Photometrics Prime 95B camera) with Zeiss Plan Apochromat 40 x/1.2 W objective and 488-nm laser excitation line.

Image series (videos) were recorded for 2 min unless stated otherwise from seedlings mounted in water on chambered slides as described previously [3 (link), 25 (link), 26 (link)]. SDCM recordings were performed using an inverted spinning disc confocal microscope (Yokogawa CSU-X1 on a Nikon Ti-E platform, laser box Agilent MLC400, camera Andor Ixon) with plan apochromat × 100 oil (NA = 1.45) lens, laser lines set at 488 and 561 nm, and image interval 1 s. VAEM recordings were generated using the Leica AF6000 LX fluorescence platform with integrated TIRF module and a Leica DFC350FXR2 digital camera, with plan apochromat × 100 oil (NA = 1.46) lens, 400 nm peak excitation, and 210 ms exposure time, allowing for image interval 0.5 s.

Pollen was collected from outdoor- or glasshouse-grown Nicotiana tabacum cv. Samsun flowers before opening of the flowers during warm and dry weather and kept frozen at −20°C until further use. For each biolistic pollen transformation, 1 mg of pollen grains germinating on solid pollen culture medium was used and bombardment with DNA-coated gold was performed using a particle delivery system (PDS-1000/He; Bio-Rad) at 1100 psi (Kost et al., 1998 (link)). For subcellular protein localization studies, 2–6 μg of plasmid DNA was used and 5–8 h-old germinated pollen tubes were analyzed with a spinning disk confocal microscope (Yokogawa CSU-X1 on Nikon Ti-E platform, laser box Agilent MLC400 with sCMOS camera PRIM 95B Photometrics) using a Plan Apochromat 40x WI objective and a 488 nm laser line. Time lapse series images were taken for 1 min at 2 s intervals. Camera and microscope settings were kept constant to allow for comparative imaging.