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.