Introduction
Stomata are an epidermal structure composed of a pore surrounded by a
pair of guard cells and, in certain cases, by subsidiary cells (Limaet al. 2018). Fossil records coupled to recent genomics studies
suggest that stomata exists since at least 400 million years ago, being
found from Bryophytes to Tracheophytes, with a particular loss in
Liverworts during Bryophytes evolution (Harris et al. 2020). The
appearance of stomata greatly contributed for plant adaptation to the
terrestrial environment, which is due to the fact that stomatal opening
allows the exchange of H2O and CO2between the leaf and the environment (Qu et al. 2017; Medeiroset al. 2019). More importantly, the active regulation of stomatal
movements and the faster stomatal responses found in angiosperms are
important characteristics that contribute to explain the success of this
plant group in dominating both natural and agricultural ecosystems, when
compared to plants of the basal lineage (Brodribb et al. 2019).
Furthermore, evidence suggests that slower stomata can limit the
photosynthetic rate (A ) by up to 10% (McAusland et al.2016) and faster stomatal responses can improve plant growth, yield,
water use efficiency (WUE) and drought tolerance (Papanatsiou et
al. 2019; Qu et al. 2020). Thus, understanding the mechanisms
that modulate stomatal speediness is important to comprehend the dynamic
of natural ecosystems and to breed plants towards photosynthesis and WUE
improvement (Lawson & Vialet-Chabrand 2018).
Stomata respond to a wide range of endogenous and environmental cues,
including changes in CO2 concentration, light quantity
and quality, vapour pressure deficit, and phytohormones (Gago et
al. 2020). Additionally, it has been shown that exogenous application
of sugars and genetic alteration of enzymes associated with sucrose
metabolism substantially alter stomatal conductance
(g s) (Antunes et al. 2012; Kelly et
al. 2013; Lugassi et al. 2015; Li et al. 2016; Dalosoet al. 2016b; Antunes et al. 2017; Medeiros et al.2018; Kelly et al. 2019; Flütsch et al. 2020b; Freireet al. 2021). These studies strengthen the idea that
mesophyll-derived metabolites have a great influence on the regulation
of stomatal movement (Mott 2009; Fujita et al. 2019). Notably,
reduced sugar import into guard cells compromisesg s, A and plant growth (Antunes et
al. 2017) and substantially reduces stomatal speediness (Flütschet al. 2020a). On the other hand, exogenous application of high
concentrations of sucrose induce stomatal closure in tomato (Kellyet al. 2013), Arabidopsis (Medeiros et al. 2018) and
plants of the basal lineage (Kottapalli et al. 2018). Thus,
sucrose seems to be an important metabolic signal that coordinates theA -g s trade-off (Flütsch & Santelia 2021).
However, despite recent advances in our understanding of the regulation
of stomatal speediness in model angiosperm species (Lawson &
Vialet-Chabrand 2018), it remains unclear whether these mechanisms are
also present in plants of the basal lineage and, if so, whether they
contribute to explain the slower stomatal responses found in ferns, when
compared to angiosperms.
Ferns and angiosperms share a common genetic machinery that controls
both stomatal formation and stomatal responses to light,
CO2 and abscisic acid (ABA) (Sussmilch et al.2017, 2019; Harris et al. 2020). However, whether ferns are able
to respond to endogenous ABA is extensively debated. The
ABA-responsiveness of ferns has been supported by studies in which the
magnitude of ABA-induced stomatal closure is very low (Ruszala et
al. 2011; Hõrak et al. 2017) and/or the ABA concentration used
is too high (e.g. 100 µM) (Plackett et al. 2021). On the other
side, evidences suggest that hydraulic and osmotic mechanisms
independent of ABA are the major drivers of stomatal closure in ferns
(Cardoso et al. 2019; Cardoso & McAdam 2019). Indeed, it was
recently shown that ferns and lycophyte species lack ABA-responsiveness
but an osmotic-mediated stomatal closure mechanism is likely found in
these species (Gong et al. 2021). This hypothesis is further
supported by previous results suggesting that sucrose-induced stomatal
closure is a conserved mechanism throughout land plant evolution
(Kottapalli et al. 2018). However, the last two works were
performed using extremely high concentrations of sucrose (200 mM) and
sorbitol (800 mM). It is unclear therefore whether these results have
any physiological relevance (McAdam et al. 2021), especially in
ferns that have low both photosynthetic rate (Tosens et al. 2016;
Gago et al. 2019) and capacity to produce ABA (Cardoso & McAdam
2019). It is clear therefore that further studies using relevant
physiological concentration of ABA and osmotic compounds are needed to
better understand the evolution of stomatal movements regulation.
Despite the controversies regarding the ABA-responsiveness of ferns, it
is known that these plants are able to respond to blue light and changes
in vapour pressure deficit (VPD) and CO2 concentration
(reviewed in McAdam and Sussmilch 2021). However, ferns in general have
lower g s values and their stomatal responses are
much slower than those observed in angiosperms (Franks & Britton-Harper
2016; Gago et al. 2019). In this vein, we have recently shown
that the faster high CO2-induced stomatal closure found
in angiosperms compared to ferns is positively correlated with leaf
sucrose content (Lima et al. 2019). Our previous results further
suggest that ferns have higher investment of the daily
CO2 assimilated to the synthesis of metabolites related
to secondary as opposed to primary metabolism, when compared to
angiosperms (Lima et al. 2019). Considering that increased content of
flavonols, a class of secondary metabolites, leads to lower stomatal
aperture and slower stomatal closure responses (Watkins et al.2014, 2017), we hypothesize that the lower g svalues found in ferns is due to a higher investment to the synthesis of
secondary rather than primary metabolites throughout the diel course, as
compared to angiosperms. Furthermore, given that sucrose is negatively
correlated with g s (Gago et al. 2016) and
is likely involved in the regulation of stomatal movement throughout
land plant evolution (Kottapalli et al. 2018; Lima et al.2019), we further hypothesize that sucrose-mediated stomatal closure
mechanisms are important to regulate both the magnitude of stomatal
opening throughout the diel course and the differential stomatal closure
speediness between ferns and angiosperms. To test these hypotheses, we
investigated the effect of exogenous application of sucrose ong s kinetics in two ferns and a representative
angiosperm species and carried out a liquid chromatography mass
spectrometry (LC-MS)-based metabolic fingerprinting analysis, that
mostly detect secondary metabolites (Perez de Souza et al. 2021),
in leaves from two ferns and two angiosperms species harvested
throughout the diel course.