2.4 Water and dye uptake experiments
We first monitored epidermal transpiration in ten fully expanded leaves by letting them dry with their petiole covered with parafilm at room temperature (average 23±3ºC) and 30±3% of relative humidity (RH), weighing them every two hours with a four-digit precision scale. We then divided the weights by the leaf projected area (x2), fitted the temporal loss of each leaf weight to a linear regression function for the initial 10 hours, and averaged the slopes. This value served as a reference of epidermal evaporation from both leaf sides (slope = -0.041 mg cm-2 h-1, averager 2=0.95). After that, we calculated the relative leaf water content (RWC) using the formula RWC=[(FWt-DW)/(FW0-DW)], were FWt is the fresh weight at time t, DW is the dry weight, and FW0 is the fresh weight at time 0 (Figure 5a). We further calculated the initial water content of the leaves (Wi) as the difference between the fresh weight minus the dry weight per unit leaf area.
To estimate maximum rates of foliar uptake, five leaves were weighed (Wt0) before being submerged in distilled water, with the petiole sealed, but not submerged, for 15 min (wet cycle ). To eliminate any surface water, these leaves were centrifuged at 1800 rpm for 2 min using a Sorvall RC6 Ultracentrifuge, and immediately weighed with a precision scale (Wt1). Leaves were left to dry at room temperature for 15min (dry cycle ) and weighed again before immersion (Wt2). The cycle was repeated five times.
To understand the possible contribution of each surface to foliar water uptake, five leaves were loaded with 350 µL of distilled water (350 mg) evenly distributed in 35 droplets (10 µL each) covering roughly 25-30% of one surface, five leaves loaded on the abaxial side, and five loaded on the adaxial surface. When the droplets disappeared from the surfaces, the leaves were weighed with a precision scale, loaded again with the same amount of water, and this cycle repeated. Droplet disappearance was faster from the abaxial leaf surface, resulting in more time points during the 10 hours of the experiment.
To calculate water absorbed by each surface, we used the following formula for each cycle
ABSt= [(Wt – Wt0) – (EPt LA*(t-t0))]/ LA, (1),
where ABSt is the water absorbed, measured in mg cm-2, at time t (after droplet disappearance from the surfaces), Wt is the weight of the leaves in mg, LA* the leaf exposed area (the projected area of the unloaded surface plus 70% of the loaded one), LA is the projected leaf area (only one surface) measured in cm2, and Wt0 the initial weight of the leaves in mg. EPt is the epidermal transpiration rate of the leaf surface not covered by droplets (one side plus 70% of the other), which assumes that water loss from the two sides occurs at the same rate: EPt= -0.035 mg cm-2h-1.
To understand the pathways of water uptake, we used a 1% aqueous solution of the apoplastic fluorescent dye tracer Lucifer Yellow (LY; CH dilithium salt; Sigma). Dye (10 µL droplets) was applied to either the adaxial or the abaxial surfaces within a humidified chamber. We waited until the droplets disappeared from the surface, and then used a paper tissue to wipe any traces of the dye from the leaf surface prior to making transverse sections with a double edge razor blade. Sections were mounted in an aqueous solution of 70% glycerol, and immediately observed with a Zeiss LSM700 Confocal Microscope (wavelength 488nm). Similar sections of the same leaves, but in areas without the dye, were used as negative controls.
We further evaluated the rate of water and dye uptake by each leaf sidein vivo , applying 5 µL droplets to the leaves of a seedling, and then taking images every five seconds (Videos S1-S4).