Ferns did not respond to ABA and display slower mannitol
and sucrose induced stomatal closure
We next investigated the effect of exogenous application of ABA (5 µM),
sucrose (25 mM) or mannitol (25 mM) on stomatal closure kinetics of the
two ferns and in V. unguiculata . Given the similarity of both
metabolic and stomatal responses throughout the diel course between both
angiosperms species (Lima et al. 2019), the stomatal closure
kinetics were carried out using only one representative angiosperm
species (V. unguiculata ). Ferns stomata did not respond to ABA
(Figures 2a,d). However, exogenous application of both mannitol and
sucrose decreased g s in all plants (Figures
2b,c), with no differences in the relative decrease ing s between ferns and the angiosperm observed
following the provision of sucrose and mannitol (Figures 2e,f).
The stomatal responses of the ferns to mannitol and the response of the
fern P. aureum to sucrose were best fitted into a linear model,
while the stomatal response of the fern M. scolopendria to
sucrose and all stomatal responses of the angiosperm V.
unguiculata reached steady state and were best fitted using an
exponential decay model (Figures S2-S4). We next investigated the
acceleration observed during stomatal closure by curve fitting the data
using non-linear regressions. Linear responses exhibit constant speed
and thus zero acceleration. With exception of the response of M.
scolopendria to sucrose, the acceleration of the other treatments in
ferns is zero. This approach also determines the plateau (in seconds) in
which the acceleration starts to decrease. The plateau of theg s response to sucrose was achieved at 2915 and
3681 seconds in V. unguiculata and the fern M.
scolopendria , respectively. This analysis suggests that the stomatal
response to sucrose is faster in V. unguiculata than M.
scolopendria , as evidenced by the shorter time needed to reach the
plateau. The plateau of V. unguiculata g sresponse to ABA and mannitol was observed at 3461 and 3920 seconds,
respectively (Figures S2-S4). The data was subsequently transformed
following a maximum-minimum normalization (0-1 range) to allow
time-series comparison. The results highlight that the sucrose-induced
stomatal closure of the angiosperm was unique to reachVmax (Cluster 1), as compared to the other
treatments, which were clustered separately (Figure S5).
We further analysed the slope of the stomatal closure kinetics, a
parameter commonly used to estimate stomatal speediness (McAuslandet al. 2016). Considering that the ferns used here did not
respond to ABA in the time period analysed, the slope of stomatal
closure kinetics under ABA was higher in V. unguiculata than both
ferns. The results further highlight that ferns have slowerg s responses to mannitol than the angiosperm,
whilst no statistical difference in the slope of stomatal closure
kinetics induced by sucrose between ferns and angiosperms was observed
(Figures 3a-c). We next compared these values with those previously
reported during stomatal closure induced by dark or high
CO2 concentration (Lima et al. 2019). The fastest
stomatal closure of V. unguiculata was observed under ABA and
high CO2, while these signals represent the slower
stomatal closure in the fern P. aureum . No statistical difference
among stomatal closure signals in the fern M. scolopendria was
observed. Interestingly, the speed of stomatal closure induced by
mannitol and sucrose are among the lowest in V. unguiculata ,
while sucrose-induced stomatal closure represents the higher average
value in both ferns (Figures 3d-f). Taken together, these results
highlight that an osmotic-mediated mechanism of stomatal closure is
conserved among ferns and angiosperms and that the slower stomatal
closure in ferns is associated to their limited capacity to respond to
ABA and mesophyll-derived sucrose.