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.