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).