Stomatal Blue Light Response Is Present in Early Vascular Plants

Light is a major environmental factor required for stomatal opening. Blue light (BL) induces stomatal opening in higher plants as a signal under the photosynthetic active radiation. The stomatal BL response is not present in the fern species of Polypodiopsida. The acquisition of a stomatal BL response might provide competitive advantages in both the uptake of CO₂ and prevention of water loss with the ability to rapidly open and close stomata. We surveyed the stomatal opening in response to strong red light (RL) and weak BL under the RL with gas exchange technique in a diverse selection of plant species from euphyllophytes, including spermatophytes and monilophytes, to lycophytes. We showed the presence of RL-induced stomatal opening in most of these species and found that the BL responses operated in all euphyllophytes except Polypodiopsida. We also confirmed that the stomatal opening in lycophytes, the early vascular plants, is driven by plasma membrane proton-translocating adenosine triphosphatase and K⁺ accumulation in guard cells, which is the same mechanism operating in stomata of angiosperms. These results suggest that the early vascular plants respond to both RL and BL and actively regulate stomatal aperture. We also found three plant species that absolutely require BL for both stomatal opening and photosynthetic CO₂ fixation, including a gymnosperm, C. revoluta, and the ferns Equisetum hyemale and Psilotum nudum.Stomata regulate gas exchange between plants and the atmosphere (Zeiger, 1983; Assmann, 1993; Roelfsema and Hedrich, 2005; Shimazaki et al., 2007; Kim et al., 2010). Acquisition of stomata was a key step in the evolution of terrestrial plants by allowing uptake of CO₂ from the atmosphere and accelerating the provision of nutrients via the transpiration stream within the plant (Hetherington and Woodward, 2003; McAdam and Brodribb, 2013). Stomatal aperture is regulated by changes in the turgor of guard cells, which are induced by environmental factors and endogenous phytohormones. Light is a major factor in the promotion of stomatal opening, and the opening is mediated via two distinct light-regulated pathways that are known as photosynthesis- and blue light (BL)-dependent responses under photosynthetic active radiation (PAR; Vavasseur and Raghavendra, 2005; Shimazaki et al., 2007; Lawson et al., 2014).The photosynthesis-dependent stomatal opening is induced by a continuous high intensity of light, and the action spectrum for the stomatal opening resembles that of photosynthetic pigments in leaves (Willmer and Fricker, 1996). Both mesophyll and guard cells possess photosynthetically active chloroplasts, and their photosynthesis has been suggested to contribute to stomatal opening in leaves. The decrease in the concentration of intercellular CO₂ (Ci) caused by photosynthetic CO₂ fixation or some unidentified mediators and metabolites from mesophyll cells is supposed to elicit stomatal opening, although the exact nature of the events is unclear (Wong et al., 1979; Vavasseur and Raghavendra, 2005; Roelfsema et al., 2006; Mott et al., 2008; Lawson et al., 2014).BL-dependent stomatal opening requires a strong intensity of PAR as a background: weak BL solely scarcely elicits stomatal opening, but the same intensity of BL induces the fast and large stomatal opening in the presence of strong red light (RL; Ogawa et al., 1978; Shimazaki et al., 2007). Since such stomatal opening requires BL under the RL or PAR, we call the opening reaction a BL-dependent response of stomata. BL-dependent stomatal response takes place and proceeds in natural environments because the sunlight contains both BL and RL and facilitates photosynthetic CO₂ fixation (Assmann, 1988; Takemiya et al., 2013a). In this stomatal response, BL and PAR (BL, RL, and other wavelengths of light) seem to act as a signal and an energy source, respectively.The BL-dependent stomatal opening is initiated by the absorption of BL by phototropin1 and phototropin2 (Kinoshita et al., 2001), the plant-specific BL receptors, in guard cells followed by activation of the plasma membrane proton-translocating adenosine triphosphatase (H+-ATPase; Kinoshita and Shimazaki, 1999). Two newly identified proteins, protein phosphatase1 and blue light signaling1 (BLUS1), mediate the signaling between phototropins and H+-ATPase (Takemiya et al., 2006, 2013a, 2013b). The activated H+-ATPase evokes a plasma membrane hyperpolarization, which drives K⁺ uptake through the voltage-gated, inward-rectifying K⁺ channels (Assmann, 1993; Shimazaki et al., 2007; Kim et al., 2010; Kollist et al., 2014). The accumulation of K⁺ causes water uptake and increases turgor pressure of guard cells, and finally results in stomatal opening. The BL-dependent opening is enhanced by RL, and BL at low intensity is effective in the presence of RL (Ogawa et al., 1978; Iino et al., 1985; Shimazaki et al., 2007; Suetsugu et al., 2014). These stomatal responses by RL and BL are commonly observed in a number of seed plants so far examined.Fine control of stomatal aperture to various environmental factors has been characterized in many angiosperms. Although morphological and mechanical diversity of stomata is widely documented, little is known about the functional diversity (Willmer and Fricker, 1996; Hetherington and Woodward, 2003). Our previous study indicated that BL-dependent stomatal response is absent in the major fern species of Polypodiopsida, including Adiantum capillus-veneris, Pteris cretica, Asplenium scolopendrium, and Nephrolepis auriculata, but the stomata of these species open by PAR including RL (Doi et al., 2006). When the epidermal peels isolated from A. capillus-veneris are treated with photosynthetic electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1dimethylurea (Doi and Shimazaki, 2008), the response is completely inhibited, but the responses in the seed plants of Vicia faba and Commelina communis are relatively insensitive to 3-(3,4-dichlorophenyl)-1,1dimethylurea (Schwartz and Zeiger, 1984). These findings suggest that there is functional diversity in light-dependent stomatal response in different lineages of land plants. In accord with this notion, the different sensitivities of stomatal response to abscisic acid and CO₂ have been reported among the plant species of angiosperm, gymnosperm, ferns, and lycophytes (Mansfield and Willmer, 1969; Brodribb and McAdam, 2011), although the exact responsiveness to abscisic acid and CO₂ is still debated (Chater et al., 2011, 2013; Ruszala et al., 2011; McAdam and Brodribb, 2013).To address the origin and distribution of stomatal light responses, we investigated the presence of a stomatal response using a gas exchange method and various lineages of vascular plants, including euphyllophytes and lycophytes. Unexpectedly, all plant lineages except Polypodiopsida in monilophytes exhibited a stomatal response to BL in the presence of RL, suggesting that the response was present in the early evolutionary stage of vascular plants. We also report the stomatal opening in response to RL in these plant species.