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.