The Stomata of the Fern Adiantum capillus-veneris Do Not Respond to CO2 in the Dark and Open by Photosynthesis in Guard Cells  

Abstract

The stomata of the fern Adiantum capillus-veneris lack a blue light-specific opening response but open in response to red light. We investigated this light response of Adiantum stomata and found that the light wavelength dependence of stomatal opening matched that of photosynthesis. The simultaneous application of red (2 mmol m 22 s 21 ) and far-red (50 mmol m 22 s 21 ) light synergistically induced stomatal opening, but application of only one of these wavelengths was ineffective. Adiantum stomata did not respond to CO 2 in the dark; the stomata neither opened under a low intercellular CO 2 concentration nor closed under high intercellular CO 2 concentration. Stomata in Arabidopsis (Arabidopsis thaliana), which were used as a control, showed clear sensitivity to CO 2 . In Adiantum, stomatal conductance showed much higher light sensitivity when the light was applied to the lower leaf surface, where stomata exist, than when it was applied to the upper surface. This suggests that guard cells likely sensed the light required for stomatal opening. In the epidermal fragments, red light induced both stomatal opening and K 1 accumulation in guard cells, and both of these responses were inhibited by a photosynthetic inhibitor, 3-(3,4-dichlorophenyl)-1, 1-dimethylurea. The stomatal opening was completely inhibited by CsCl, a K 1 channel blocker. In intact fern leaves, red lightinduced stomatal opening was also suppressed by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. These results indicate that Adiantum stomata lack sensitivity to CO 2 in the dark and that stomatal opening is driven by photosynthetic electron transport in guard cell chloroplasts, probably via K 1 uptake.

Evolution of the Stomatal Regulation of Plant Water Content

“…Indeed, the presence of chloroplasts in guard cells appears to be an ancestral trait, with distinctive coloring of the guard cells in comparison to epidermal pavement cells noted in rhyniophytoids (Edwards et al, 1998), suggesting that these earliest fossilized stomata similarly contained chloroplasts. While in angiosperms photosynthesis in the mesophyll appears to provide a major signal for driving stomatal opening in the light (Roelfsema et al, 2002;Lawson et al, 2014), in basal vascular plants, stomatal opening in the light can be driven by guard cell-autonomous photosynthesis alone, with rapid stomatal opening occurring in the isolated epidermis of leptosporangiate ferns (McAdam and Brodribb, 2012), which lack stomatal responses to blue light and hence open only by photosynthetic processes (Doi et al, 2006(Doi et al, , 2015Doi and Shimazaki, 2008). These data indicate that a mesophyll signal for stomatal opening in the light likely evolved after the divergence of seed plants (McAdam and Brodribb, 2012).…”

“…The apparent involvement of the H + -ATPase in the stomatal opening of mosses appears to confirm a common mechanism with vascular plants , although more work in bryophytes is required to confirm this potentially ancient mechanism. Extant lycophyte and fern stomata have the capacity to open rapidly in the light (Mansfield and Willmer, 1969;Doi and Shimazaki, 2008;McAdam and Brodribb, 2012), with lycophyte stomata possessing both photosynthetically driven stomatal opening in red light-as well as blue light-triggered opening, while leptosporangiate ferns (which comprise more than 96% of fern specific diversity; Palmer et al, 2004) have lost blue light stomatal signaling (Doi et al, 2015). The reason behind this loss of blue light stomatal signaling in derived ferns is, as yet, unknown, but may be due to a chimeric red-blue photoreceptor, an adaptation associated with photosynthesis in low light environments (Kawai et al, 2003).…”

“…A common feature of all studies in this area is a conserved tendency to respond to CO 2 , particularly low CO 2 , which likely reflects a common photosynthetic signaling in all stomata Franks and Britton-Harper, 2016). However, major differences across lineages in the way stomata instantaneously respond to CO 2 in the dark (Doi and Shimazaki, 2008; as well as the influence of ABA on the sensitivity of stomata to CO 2 reflect as yet undescribed and potentially important mechanistic differences in the way stomata respond to more natural endogenous and environmental signals. Further work in this field is required to reveal these key mechanistic differences.…”

Fern and Lycophyte Guard Cells Do Not Respond to Endogenous Abscisic Acid

“…Several major discoveries have demonstrated the power of a phylogenetic approach in the investigation of stomatal physiology, including evidence for absent phototropinmediated responses of guard cells to blue light in the basal lineages of vascular plants (Doi et al, 2006;Doi and Shimazaki, 2008) and, more recently, evidence that the stomata of earlybranching vascular plants are only equipped with very simple responses to light and water content Brodribb and McAdam, 2011;Haworth et al, 2011). These studies suggest that during the 400 million years since the evolution of stomata (Edwards et al, 1998), the control of stomatal aperture has increased in complexity and that this is reflected in the characteristic stomatal behaviors of the different clades of living vascular plants (McAdam and Brodribb, 2012).…”

Perforated-tape Epidermal Detachment (PED): A simple and rapid method for isolating epidermal peels from specific areas of Arabidopsis leaves

“…For the conventional blender method, approximately 5 leaves were fractured in cold water using a Waring commercial blender (model 7011S with MC1 mini-container, FMI, Tokyo, Japan) and then filtered through nylon mesh (N-No. 305T, 48 µm, NBC, Tokyo, Japan) to collect the fragmented tissues, as previously described (Allen et al 1999;Doi and Shimazaki 2008;Ichida et al 1997). For PED, Time Tape (Hirasawa, Tokyo, Japan) was used to hold the adaxial surface of the leaf, and Scotch Magic Tape (3M, Tokyo, Japan; referred to as Scotch Tape here) perforated with a hole (see below) was used to detach the abaxial epidermis of Arabidopsis and young tobacco leaves.…”