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.