Metabolism-mediated stomatal closure mechanisms contribute
to explain the slower stomatal movements in ferns, compared to
angiosperms
The speed of stomatal closure is 2.2 and 8.4-fold lower than stomatal
opening in response to light and CO2 transitions within
ferns (Lima et al. 2019). This suggests that the mechanisms that
coordinate stomatal closure are severely impaired in ferns, as compared
to those controlling stomatal opening. Beyond morphological and genetic
differences that aid to explain the evolution toward a highly responsive
stomata in angiosperms (Cai et al. 2017; Sussmilch et al.2019; Harris et al. 2020; Gong et al. 2021), we put
forward the hypothesis that metabolism-mediated mechanisms that
coordinate the A -g s trade-off may
contribute to explain the rapid control of stomatal movements found in
angiosperms. This idea relies in the fact that the faster high
CO2-induced stomatal closure found in angiosperms was
positively correlated with leaf sucrose content (Lima et al.2019), a metabolite largely described as important to theA -g s trade-off regulation (Talbott &
Zeiger 1998; Daloso et al. 2016a; Granot & Kelly 2019; Flütsch
& Santelia 2021). This suggests that the higher photosynthetic capacity
of angiosperms (Tosens et al. 2016; Gago et al. 2019),
which results in higher sucrose production (Lima et al. 2019), is
a key facilitator of rapid closure of the stomata. Indeed, exogenous
application of sucrose and mannitol reduced g s in
both ferns, but in a lower speed, when compared to angiosperms. Although
no difference in the slope of the g s kinetic
following provision of sucrose between ferns and angiosperms was
observed (Figure 3c), this kinetic reached a maximum velocity
(Vmax ) only in V. unguiculata (Figure S5). Furthermore,
the angiosperm reached a plateau earlier than the fern M.
scolopendria , whilst the fern P. aureum did not reach a plateau
(Figures S2i, S3i and S4i). These results indicate that V.
unguiculata has a greater capacity to rapidly respond to the
accumulation of sucrose.
Interestingly, both sucrose and mannitol induced stomatal closure in
ferns and V. unguiculata (Figures 2b-c). However, the speed of
sucrose-induced stomatal closure is higher than the mannitol treatment
in all species. This is evidenced by the higher slope of stomatal
closure kinetic under sucrose in P. aureum (Figure 3e), the
plateau reached solely under sucrose treatment in M. scolopendria(Figures S2h-i), and the earlier plateau and the maximum velocity
reached in V. unguiculata under sucrose (Figures S4h-i), when
compared to the mannitol treatment within each species. These results
suggest that stomata from ferns and V. unguiculata exhibit both
osmotic and non-osmotic responses. In fact, it has been proposed that
mesophyll-derived sucrose induces stomatal closure by two different
mechanisms: (i) – by an osmotic mechanism, probably associated to the
accumulation of sucrose and other osmolytes in the apoplastic space of
guard cells (Lu et al. 1995, 1997; Kang et al. 2007a b);
and (ii) by a signalling mechanism, in which these compounds would be
perceived by guard cells and the stomatal closure triggered by
signalling transduction pathways associated to ABA and hexokinase (Kellyet al. 2013, 2019; Lugassi et al. 2015). However, recent
evidence highlights that the fern Matteuccia struthiopteris and
the lycophyte Selaginella moellendorffii lack ABA-responsiveness
due to a disruption in the ABA signalling pathway (Gong et al.2021). This work has demonstrated that these species have lower level of
ROS and exhibits no increases in both nitric oxide (NO) and
Ca+2 in their guard cells following provision of ABA
(Gong et al. 2021). Therefore, whilst our results highlight that
fern stomata do respond to exogenous application of both mannitol and
sucrose at physiologically relevant concentrations, confirming that a
metabolism-mediated stomatal closure mechanism is conserved among ferns
and angiosperms, it remains unclear whether the sucrose-induced stomatal
closure involves the hexokinase/ABA pathway described for angiosperms.
Our metabolic fingerprinting analysis suggests that the preferential
allocation of the diel course CO2 assimilated toward the
secondary metabolism may also influence stomatal movement regulation in
ferns. It has been shown that plants with higher accumulation of
secondary metabolites have reduced levels of ROS in their guard cells
(Watkins et al. 2014), which is associated to the capacity of
these metabolites in removing ROS (Watkins et al. 2017; Delfinet al. 2019). It is thus reasonable to hypothesize that the
higher allocation toward the secondary metabolism observed in ferns
would leads to lower level of ROS in their guard cells, as compared to
angiosperms (Figure 8c), which was indeed observed in a previous study
(Gong et al. 2021). Therefore, a preferential carbon allocation
toward the secondary rather than the primary metabolism would leads to
both lower g s throughout the diel course and
slower stomatal closure responses in ferns, when compared to
angiosperms.