2.5 Foliar water uptake experiment
Plants were grown in the greenhouse for a year and a half before being transferred to a climate chamber. Conditions in the climate chamber were 12-hour days, with 250 µmol m-2 s-1of light, 25/20°C day/night temperatures, and 20–35% relative humidity. Plants were acclimated to these conditions for three months before the start of the experiment. Drought was simulated by withholding water for six weeks in at least two plants per species, while at least two other plants per species were kept under a well-watered regime (i.e. thorough watering every 10 days).
For the FWU experiment, we used Lucifer Yellow carbohydrazide (LYCH), a fluorescent tracer often used to study water transport in vivo in plants (Oparka and Prior 1988; Oparka et al. 1988; Farrar et al. 1992; Bederska et al. 2012), including studies focusing on FWU (Burrows et al. 2013; Eller et al. 2013, 2016; Pina et al. 2016; Holanda et al. 2019; Losada et al. 2021). LYCH was used as an apoplastic tracer, as it is unable to cross plasma membranes. A 50mM stock solution of LYCH as dilithium salt (Sigma) was prepared in dH2O and stored at −18°C. Before use, the stock solution was thawed and diluted in dH2O to a working solution of 0.5 mM.
The evening before the experiment, leaves from both treatments (i.e. drought and well-watered) were carefully excised from the plants and the cut end was sealed with an ethyl cyanoacrylate-based glue (Super Glue, Loctite) to prevent both water loss and tracer uptake during the experiment. Rudimental humidity chambers were assembled by laying out wet paper towels inside plant propagators with transparent covers. The excised leaves were balanced on inverted Petri dishes, so that they would not touch the wet towels. On the day of the experiment, 10–100 μl droplets (depending on the species) of LYCH solution were applied to the area of interest of each leaf (see Table 2 ) at 8:00 a.m. (local time), two hours into the light period (250 µmol m-2s-1, 25°C), to simulate morning fog and dew. Three hours after the application of the tracer, the lamina of the leaves was rinsed thoroughly three times by immersion in dH2O to remove the residual surface dye and carefully dried with paper towel. Free-hand sections were made of the treated leaf areas and mounted in 9:1 glycerol:PBS to minimize efflux and redistribution of the dyes (Mastroberti and de Araujo Mariath 2008). We made additional free-hand sections of untreated leaves (i.e. controls) to observe the natural autofluorescence of leaf tissues. All leaves were imaged immediately after sectioning and mounting in a fluorescence stereo microscope fitted with a digital camera (M205FA, Leica Microsystems), using both the GFP2 (ex. 480/40 nm; em. 510 nm LP) and the ET GFP (ex. 470/40 nm; em. 525/50 nm) filter sets.