Stomatal pores, each surrounded by a pair of guard cells, regulate CO2 uptake and water loss from leaves. Stomatal opening is driven by the accumulation of K+ salts and sugars in guard cells, which is mediated by electrogenic proton pumps in the plasma membrane and/or metabolic activity. Opening responses are achieved by coordination of light signaling, light-energy conversion, membrane ion transport, and metabolic activity in guard cells. In this review, we focus on recent progress in blue- and red-light-dependent stomatal opening. Because the blue-light response of stomata appears to be strongly affected by red light, we discuss underlying mechanisms in the interaction between blue-light signaling and guard cell chloroplasts.

The stomatal pores of higher plants allow for gaseous exchange into and out of leaves. Situated in the epidermis, they are surrounded by a pair of guard cells which control their opening in response to many environmental stimuli, including blue light. Opening of the pores is mediated by K(+) accumulation in guard cells through a K(+) channel and driven by an inside-negative electrical potential. Blue light causes phosphorylation and activation of the plasma membrane H(+)-ATPase that creates this potential. Thus far, no blue light receptor mediating stomatal opening has been identified, although the carotenoid, zeaxanthin, has been proposed. Arabidopsis mutants deficient in specific blue-light-mediated responses have identified four blue light receptors, cryptochrome 1 (cry1), cryptochrome 2 (cry2), phot1 and phot2. Here we show that in a double mutant of phot1 and phot2 stomata do not respond to blue light although single mutants are phenotypically normal. These results demonstrate that phot1 and phot2 act redundantly as blue light receptors mediating stomatal opening.

Phototropins are autophosphorylating protein kinases of plantspecific blue light receptors. They regulate various blue light responses, including phototropism, chloroplast movements, hypocotyl growth inhibition, leaf flattening, and stomatal opening. However, the physiological role of autophosphorylation remains unknown. Here, we identified phosphorylation sites of Ser or Thr in the N terminus, Hinge1 region, kinase domain, and C terminus in Arabidopsis phototropin1 (phot1) by liquid chromatographytandem mass spectrometry in vivo. We substituted these Ser or Thr residues with Ala in phot1 and analyzed their functions by inspecting the phot1-mediated responses of stomatal opening, phototropism, chloroplast accumulation, and leaf flattening after the transformation of the phot1 phot2 double mutant. Among these sites, we found that autophosphorylation of Ser-851 in the activation loop of the kinase domain was required for the responses mentioned above, whereas the phosphorylation of the other Ser and Thr, except those in the activation loop, was not. Ser-849 in the loop may have an additional role in the responses. Immunological analysis revealed that Ser-851 was phosphorylated rapidly by blue light in a fluence-dependent manner and dephosphorylated gradually upon darkness. We conclude that autophosphorylation of Ser-851 is a primary step that mediates signaling between photochemical reaction and physiological events.protein kinase ͉ stomata ͉ photoreceptor ͉ light signaling P hototropins (phot1 and phot2) mediate multiple blue light responses in Arabidopsis, including phototropism, chloroplast movements, leaf flattening, leaf positioning, stomatal opening, and rapid inhibition of hypocotyl growth (1-5). These responses enhance photosynthesis and optimize plant growth, particularly under weak light (6). Under strong light, phot2 induces a chloroplast-avoidance response to prevent photodamage to photosynthetic machinery (7). Phototropins, therefore, are essential proteins for the survival of higher plants and the extension of their living areas, particularly under ever-changing environments of light.Phototropins are blue light receptor protein kinases with two light, oxygen, voltage (LOV) domains in the N terminus and a Ser/Thr protein kinase in the C terminus (8). The LOV domains possess noncovalent binding sites for the chromophore flavin mononucleotide (FMN) and cysteine residues (2, 9-11) and produce a covalent cysteinyl adduct with FMN when LOVs are illuminated with blue light (12, 13). The cysteinyl adduct formation is the primary photochemical process; the adduct formation induces conformational changes in the LOV2 domain (14, 15) and J␣-helix (16) and leads to phototropin phosphorylation and subsequent physiological processes (17, 18). Phototropin phosphorylation by blue light is demonstrated to be autophosphorylation through the use of recombinant proteins of PHOT1 and PHOT2 expressed in insect cells (2, 9, 17).Phototropin was initially found as a plasma membraneassociated phosphorylated protein in etiolate...

Phototropins (phot1 and phot2) are plant-specific blue light receptors for phototropism, chloroplast movement, leaf expansion, and stomatal opening. All these responses are thought to optimize photosynthesis by helping to capture light energy efficiently, reduce photodamage, and acquire CO 2 . However, experimental evidence for the promotion of plant growth through phototropins is lacking. Here, we report dramatic phototropin-dependent effects on plant growth. When plants of Arabidopsis thaliana wild type, the phot1 and phot2 mutants, and the phot1 phot2 double mutant were grown under red light, no significant growth differences were observed. However, if a very low intensity of blue light (0.1 mmol m ÿ2 s ÿ1 ) was superimposed on red light, large increases in fresh weight up to threefold were found in those plants that carried functional PHOT1 genes. When the intensity of blue light was increased to 1 mmol m ÿ2 s ÿ1 , the growth enhancement was also found in the phot1 single mutant, but not in the double mutant, indicating that phot2 mediated similar responses as phot1 with a lower sensitivity. The effects occurred under low photosynthetically active radiation in particular. The wellknown physiological phototropin-mediated responses, including chloroplast movement, stomatal opening, and leaf expansion, in the different lines tested indicated an involvement of these responses in the blue light-induced growth enhancement. We conclude that phototropins promote plant growth by controlling and integrating a variety of responses that optimize photosynthetic performance under low photosynthetically active radiation in the natural environment.

Appropriate leaf positioning is essential for optimizing photosynthesis and plant growth. However, it has not been elucidated how green leaves reach and maintain their position for capturing light. We show here the regulation of leaf positioning under blue light stimuli. When 1-week-old Arabidopsis seedlings grown under white light were transferred to red light (25 micromol m(-2) s(-1)) for 5 d, new petioles that appeared were almost horizontal and their leaves were curled and slanted downward. However, when a weak blue light from above (0.1 micromol m(-2) s(-1)) was superimposed on red light, the new petioles grew obliquely upward and the leaves were flat and horizontal. The leaf positioning required both phototropin1 (phot1) and nonphototropic hypocotyl 3 (NPH3), and resulted in enhanced plant growth. In an nph3 mutant, neither optimal leaf positioning nor leaf flattening by blue light was found, and blue light-induced growth enhancement was drastically reduced. When blue light was increased from 0.1 to 5 micromol m(-2) s(-1), normal leaf positioning and leaf flattening were induced in both phot1 and nph3 mutants, suggesting that phot2 signaling became functional and that the signaling was independent of phot1 and NPH3 in these responses. When plants were irradiated with blue light (0.1 micromol m(-2) s(-1)) from the side and red light from above, the new leaves became oriented toward the source of blue light. When we transferred these plants to both blue light and red light from above, the leaf surface changed its orientation to the new blue light source within a few hours, whereas the petioles initially were unchanged but then gradually rotated, suggesting the plasticity of leaf positioning in response to blue light. We showed the tissue expression of NPH3 and its plasma membrane localization via the coiled-coil domain and the C-terminal region. We conclude that NPH3-mediated phototropin signaling optimizes the efficiency of light perception by inducing both optimal leaf positioning and leaf flattening, and enhances plant growth.

Phototropins are blue-light (BL) receptor serine (Ser)/threonine kinases, and contain two light, oxygen, and voltage (LOV) domains, and are members of the PAS domain superfamily. They mediate phototropism, chloroplast movement, leaf expansion, and stomatal opening of higher plants in response to BL. In stomatal guard cells, genetic analysis has revealed that phototropins mediate activation of the plasma membrane H ϩ -ATPase by phosphorylation and drive stomatal opening. However, biochemical evidence for the involvement of phototropins in the BL response of stomata is lacking. Using guard cell protoplasts, we showed that broad bean (Vicia faba) phototropins (Vfphots) were phosphorylated by BL, and that this phosphorylation of Vfphots reached to the maximum level earlier than that of the H ϩ -ATPase. Phosphorylation of both Vfphots and H ϩ -ATPase showed similar sensitivity to BL and were similarly suppressed by protein kinase and flavoprotein inhibitors. We found that a 14-3-3 protein was bound to Vfphots upon phosphorylation, and this binding occurred earlier than the H ϩ -ATPase phosphorylation. Vfphots (Vfphot1a and Vfphot1b) were expressed in Escherichia coli, and phosphorylation sites were determined to be Ser-358 for Vfphot1a and Ser-344 for Vfphot1b, which are localized between LOV1 and LOV2. We conclude that Vfphots act as BL receptors in guard cells and that phosphorylation of a Ser residue between LOV1 and LOV2 and subsequent 14-3-3 protein binding are likely to be key steps of BL response in stomata. The binding of a 14-3-3 protein to Vfphot was found in etiolated seedlings and leaves in response to BL, suggesting that this event was common to phototropin-mediated responses.Stomatal pores surrounded by a pair of guard cells in the epidermis regulate gas exchange between leaves and the atmosphere and allow CO 2 entry for both photosynthesis and the transpirational stream in higher plants (Zeiger, 1983; Assmann, 1993). Stomata open through the activation of a H ϩ pump in guard cells in response to blue light (BL; Assmann et al., 1985; Shimazaki et al., 1986). The BL-activated pump creates an inside-negative, electrical potential across the plasma membrane and drives K ϩ uptake through voltage-gated K ϩ channels (Hedrich and Schroeder, 1989; Assmann and Shimazaki, 1999; Schroeder et al., 2001). The H ϩ pump has been demonstrated to be the plasma membrane H ϩ -ATPase and is activated via phosphorylation of its C terminus with concomitant binding of the 14-3-3 protein (Kinoshita and Shimazaki, 1999; Emi et al., 2001; Palmgren, 2001). Although physiological responses downstream of BL perception have been elucidated extensively in guard cells, the mechanism of BL perception itself, an initial event in the BL response of stomata, had yet to be elucidated. Recently, genetic analysis of Arabidopsis mutants strongly suggests that phototropins (Atphot1 and Atphot2) function as BL receptors in a redundant manner and mediate BL-dependent stomatal opening (Kinoshita et al., 2001), although the biochemical eviden...

Stomata open in response to light and close after exposure to abscisic acid (ABA). They regulate gas exchange between plants and the atmosphere, enabling plants to adapt to changing environmental conditions. ABA binding to receptors initiates a signaling cascade that involves protein phosphorylation. We show that ABA induced the phosphorylation of three basic helix-loop-helix (bHLH) transcription factors, called AKSs (ABA-responsive kinase substrates; AKS1, AKS2, and AKS3), in Arabidopsis guard cells. In their unphosphorylated state, AKSs facilitated stomatal opening through the transcription of genes encoding inwardly rectifying K⁺ channels. aks1aks2-1 double mutant plants showed decreases in light-induced stomatal opening, K⁺ accumulation in response to light, activity of inwardly rectifying K⁺ channels, and transcription of genes encoding major inwardly rectifying K⁺ channels without affecting ABA-mediated stomatal closure. Overexpression of potassium channel in Arabidopsis thaliana 1 (KAT1), which encodes a major inwardly rectifying K⁺ channel in guard cells, rescued the phenotype of aks1aks2-1 plants. AKS1 bound directly to the promoter of KAT1, an interaction that was attenuated after ABA-induced phosphorylation. The ABA agonist pyrabactin induced phosphorylation of AKSs. Our results demonstrate that the AKS family of bHLH transcription factors facilitates stomatal opening through the transcription of genes encoding inwardly rectifying K⁺ channels and that ABA suppresses the activity of these channels by triggering the phosphorylation of AKS family transcription factors.

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