Plant material and growth conditions
We studied two ferns Microsorum scolopendria (Burman) Copel. andPhlebodium aureum (L.) J. Sm. and two angiosperm speciesVigna unguiculata (L.) Walp. and Nicotiana tabacum L. Both ferns were obtained at the sporophyte stage from commercial suppliers and the species was confirmed by a taxonomist (professor Dr. Alexandre Salino, Federal University of Minas Gerais, Belo Horizonte, Brazil).V. unguiculata and N. tabacum were germinated in petri dishes and the seedlings were transferred to a hydroponic system containing Hoagland´s solution (Hoagland & Arnon 1950), as previously described (Lima et al. 2019). The plants were kept under greenhouse condition with 12 hours natural photoperiod, maximum photosynthetic photon flux density (PPFD) of 500 µmol m-2 s-1, average ambient temperature of 30 ± 4 °C and relative humidity 62 ± 2 °C.
Stomatal closure kinetic analysis
Stomatal closure kinetics were assessed as described previously (Ceciliato et al. 2019). Leaves were detached and the petiole tip immediately submerged in deionized water in a petri dish. After that, a second oblique cut was made in the petiole and transferred to a 2 mL microcentrifuge tube with deionized water. Gas exchange analysis was then initiated by using an infrared gas exchange analyser (IRGA) equipped with a 6 cm² leaf chamber (Li-6400XT, LI-COR Biosciences, Inc. Lincoln, NE, USA). The gas exchange was recorded every 10 seconds for 40 minutes under 1000 µmol photons m-2s-1. After g s stabilization, different metabolites were separately added to the tube to reach the desired final concentration as follows: ABA (5 µM), sucrose (25 mM) and mannitol (25 mM). ABA is a well-known phytohormone that induces stomatal closure at this concentration (Cai et al. 2017), which was then used to confirm whether the stomatal kinetic approach is feasible for the species used here. The sucrose concentration was selected based in the fact that sucrose transport into guard cells saturate at 25 mM (Outlaw 1995). Given that stomatal closure can also occur through an osmotic mechanism (reviewed in Lima et al. 2018), we thus included mannitol treatment to investigate the osmotic effect on stomatal closure kinetics. After the addition of ABA, sucrose or mannitol to the tube containing the detached leaf, gas exchange was recorded for further 40 minutes. In order to avoid any circadian rhythm effect, all stomatal kinetic analyses were carried out at the morning period of the day, in which a higher rate of g s is observed in these species (Lima et al. 2019).