The fern Adiantum capillus-veneris lacks stomatal responses to blue light

We investigated the responses of stomata to light in the fern Adiantum capillus-veneris, a typical species of Leptosporangiopsida. Stomata in the intact leaves of the sporophytes opened in response to red light, but they did not open when blue light was superimposed on the red light. The results were confirmed in the isolated Adiantum epidermis. The red light-induced stomatal response was not affected by the mutation of phy3, a chimeric protein of phytochrome and phototropin in this fern. The lack of a blue light-specific stomatal response was observed in three other fern species of Leptosporangiopsida, i.e. Pteris cretica, Asplenium scolopendrium and Nephrolepis auriculata. Fusicoccin, an activator of the plasma membrane H(+)-ATPase, induced both stomatal opening and H(+) release in the Adiantum epidermis. Adiantum phototropin genes AcPHOT1 and AcPHOT2 were expressed in the fern guard cells. The transformation of an Arabidopsis phot1 phot2 double mutant, which lost blue light-specific stomatal opening, with AcPHOT1 restored the stomatal response to blue light. Taken together, these results suggest that ferns of Leptosporangiopsida lack a blue light-specific stomatal response, although the functional phototropin and plasma membrane H(+)-ATPase are present in this species.     

Reversal by green light of blue light-stimulated stomatal opening in intact, attached leaves of Arabidopsis operates only in the potassium-dependent, morning phase of movement

Green light reversal of blue light-stimulated stomatal opening was discovered in isolated stomata. The present study shows that the response also occurs in stomata from intact leaves. Arabidopsis thaliana plants were grown in a growth chamber under blue, red and green light. Removal of the green light opened the stomata and restoration of green light closed them to baseline values under experimental conditions that rule out a mesophyll-mediated effect. Assessment of the response to green light over a daily time course showed that the stomatal sensitivity to green light was observed only in the morning, which coincided with the use of potassium as a guard cell osmoticum. Sensitivity to green light was absent during the afternoon phase of stomatal movement, which was previously shown to be dominated by sucrose osmoregulation in Vicia faba. Hence, the shift away from potassium-based osmoregulation in guard cells is further postulated to entail a shift from blue light to photosynthesis as the primary component of the stomatal response to light. Stomata from intact leaves of the zeaxanthin-less, npq1 mutant of Arabidopsis failed to respond to the removal or restoration of green light in the growth chamber, or to short, high fluence pulses of blue or green light. These data confirm previous studies showing that npq1 stomata are devoid of a specific blue light response. In contrast, stomata from intact leaves of phot1 phot2 double mutant plants had a reduced but readily detectable response to the removal of green light and to blue and green pulses.     

The stomata of the fern Adiantum capillus-veneris do not respond to CO2 in the dark and open by photosynthesis in guard cells

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 micromol m(-2) s(-1)) and far-red (50 micromol m(-2) s(-1)) light synergistically induced stomatal opening, but application of only one of these wavelengths was ineffective. Adiantum stomata did not respond to CO2 in the dark; the stomata neither opened under a low intercellular CO2 concentration nor closed under high intercellular CO2 concentration. Stomata in Arabidopsis (Arabidopsis thaliana), which were used as a control, showed clear sensitivity to CO2. 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+ 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+ channel blocker. In intact fern leaves, red light-induced stomatal opening was also suppressed by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. These results indicate that Adiantum stomata lack sensitivity to CO2 in the dark and that stomatal opening is driven by photosynthetic electron transport in guard cell chloroplasts, probably via K+ uptake.     

Interactions between a blue-green reversible photoreceptor and a separate UV-B receptor in stomatal guard cells

Stomatal opening exhibits two main peaks of activity in the visible range-a red peak, mediated by photosynthesis, and a blue peak, mediated by one or more blue light (BL) photoreceptors. In addition, a pronounced peak in the UV-B region has been characterized, as has a smaller UV-A peak. The BL-induced stomatal opening can be reversed by green light (GL). Here we report that UV-B-induced opening is also antagonized by GL. To determine whether UV-B is being absorbed by the BL photoreceptor or by a separate UV-B receptor, the UV-B responses of two different Arabidopsis mutants, npq1 and phot1/phot2, were tested. Both putative BL-photoreceptor mutants exhibited normal stomatal opening in response to UV-B, consistent with the existence of a separate UV-B photoreceptor. Moreover, GL failed to antagonize UV-B-induced stomatal opening in the phot1/phot2 double mutant and only partially antagonized UV-B opening in npq1. Thus, both phot1 and phot 2, as well as zeaxanthin, are required for the normal GL inhibition of UV-B. A model for a photoreceptor network that regulates stomatal opening is presented. Unlike the situation in guard cells, the UV-B bending response of Arabidopsis hypocotyls during phototropism appears to be mediated by phototropins.     

Stomata from npq1, a zeaxanthin-less Arabidopsis mutant, lack a specific response to blue light

The Arabidopsis mutant npq1, which cannot accumulate zeaxanthin because of a defective violaxanthin deepoxidase, was used to investigate the role of zeaxanthin in the stomatal response to blue light. Neither dark-adapted nor light-treated guard cells or mesophyll cells of the npq1 mutant contained detectable zeaxanthin. In contrast, wild-type guard cells had a significant zeaxanthin content in the dark and accumulated large amounts of zeaxanthin when illuminated. The well-documented red light enhancement of blue light-stimulated stomatal opening, in which increasing fluence rates of background red light result in increased response to blue light, was used to probe the specific blue light response of Arabidopsis stomata. Stomata from the npq1 mutant did not have a specific blue light response under all fluence rates of background red light tested. On the other hand, stomata from leaves of hy4 (cry 1), an Arabidopsis mutant lacking blue light-dependent inhibition of hypocotyl elongation, had a typical enhancement of the blue light response by background red light. The lack of a specific blue light response in the zeaxanthinless npq1 mutant provides genetic evidence for the role of zeaxanthin as a blue light photoreceptor in guard cells.     

Phytochrome and blue light-mediated stomatal opening in the orchid,paphiopedilum

Guard cells of the orchid genus, Paphiopedilum have been reported to lack developed chloroplasts and detectable chlorophyll a autofluorescence. Paphiopedilum stomata lack a photosynthesis-dependent opening response but have a blue light-specific opening. The present study found that low fluence rate green and red light elicited stomatal opening in Paphiopedilum and this opening was reversed by far red light, indicating the presence of a phytochrome-mediated opening response. Phytochrome-dependent, red light-stimulated opening was largest under low fluence rates and decreased to near zero as fluence rate increased. A recently discovered green light reversibility of blue light-specific stomatal opening was used to probe the properties of the blue light response in Paphiopedilum stomata. Blue light-stimulated opening was completely reversed by green light in the presence of far red light. Red light enhanced the blue light response of Paphiopedilum guard cells when given as a pretreatment or together with blue light. Analysis of guard cell pigments showed that guard cells have small amounts of chlorophyll a and b, zeaxanthin, violaxanthin, antheraxanthin and lutein. Zeaxanthin content increased in response to blue light or ascorbate and declined in the dark or under illumination in the presence of dithiothreitol, indicating the presence of an active xanthophyll cycle. Thus Paphiopedilum stomata possess both a blue light-mediated opening response with characteristics similar to species with normal chloroplast development and a novel phytochrome-mediated opening response.     

Phototropins but not cryptochromes mediate the blue light-specific promotion of stomatal conductance, while both enhance photosynthesis and transpiration under full sunlight

Leaf epidermal peels of Arabidopsis (Arabidopsis thaliana) mutants lacking either phototropins 1 and 2 (phot1 and phot2) or cryptochromes 1 and 2 (cry1 and cry2) exposed to a background of red light show severely impaired stomatal opening responses to blue light. Since phot and cry are UV-A/blue light photoreceptors, they may be involved in the perception of the blue light-specific signal that induces the aperture of the stomatal pores. In leaf epidermal peels, the blue light-specific effect saturates at low irradiances; therefore, it is considered to operate mainly under the low irradiance of dawn, dusk, or deep canopies. Conversely, we show that both phot1 phot2 and cry1 cry2 have reduced stomatal conductance, transpiration, and photosynthesis, particularly under the high irradiance of full sunlight at midday. These mutants show compromised responses of stomatal conductance to irradiance. However, the effects of phot and cry on photosynthesis were largely nonstomatic. While the stomatal conductance phenotype of phot1 phot2 was blue light specific, cry1 cry2 showed reduced stomatal conductance not only in response to blue light, but also in response to red light. The levels of abscisic acid were elevated in cry1 cry2. We conclude that considering their effects at high irradiances cry and phot are critical for the control of transpiration and photosynthesis rates in the field. The effects of cry on stomatal conductance are largely indirect and involve the control of abscisic acid levels.     

Biochemical characterization of plasma membrane H+-ATPase activation in guard cell protoplasts of Arabidopsis thaliana in response to blue light

Recent genetic analysis showed that phototropins (phot1 and phot2) function as blue light receptors in stomatal opening of Arabidopsis thaliana, but no biochemical evidence was provided for this. We prepared a large quantity of guard cell protoplasts from Arabidopsis. The immunological method indicated that phot1 was present in guard cell protoplasts from the wild-type plant and the phot2 mutant, that phot2 was present in those from the wild-type plant and the phot1 mutant, and that neither phot1 nor phot2 was present in those from the phot1 phot2 double mutant. However, the same amounts of plasma membrane H+-ATPase were found in all of these plants. H+ pumping was induced by blue light in isolated guard cell protoplasts from the wild type, from the single mutants of phototropins (phot1-5 and phot2-1), and from the zeaxanthin-less mutant (npq1-2), but not from the phot1 phot2 double mutant. Moreover, increased ATP hydrolysis and the binding of 14-3-3 protein to the H+-ATPase were found in response to blue light in guard cell protoplasts from the wild type, but not from the phot1 phot2 double mutant. These results indicate that phot1 and phot2 mediate blue light-dependent activation of the plasma membrane H+-ATPase and illustrate that Arabidopsis guard cell protoplasts can be useful for biochemical analysis of stomatal functions. We determined isogenes of the plasma membrane H+-ATPase and found the expression of all isogenes of functional plasma membrane H+-ATPases (AHA1-11) in guard cell protoplasts.     

Phototropins promote plant growth in response to blue light in low light environments

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 CO2. 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 micromol m(-2) s(-1)) 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 micromol m(-2) s(-1), 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 well-known 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.     

A transgene encoding a blue-light receptor, phot1, restores blue-light responses in the Arabidopsis phot1 phot2 double mutant

Phototropins (phot1 and phot2) are suggested to be multifunctional blue-light (BL) receptors mediating phototropism, chloroplast movement, stomatal opening, and leaf expansion. The Arabidpsis phot1 phot2 double mutant lacks all of these responses. To confirm the requirement of phototropins in BL responses, the Arabidopsis phot1 phot2 double mutant was transformed with PHOT1 cDNA and the phenotypic restoration was analysed in the transformants. It was found that all BL responses were restored, although differentially, by the transformation of the Arabidopsis phot1 phot2 double mutant with PHOT1 cDNA. The results showed that phot1 was an essential component for all these BL responses in planta, and that the cellular level of phot1 might determine the individual BL responses.     

Phytochrome involvement in stomatal movement in Pisum sativum, Vicia faba and Pelargonium sp.

Red and blue light triggered the opening of isolated stomata of Pisum sativum L. cv. Peleg Alvador, Vicia faba L. (unknown cultivar) and Pelargonium sp. The stimulatory effect of short irradiation with red or blue light was reversed by a subsequent short irradiation with far-red light. In Pisum the stimulatory effect of a continuous irradiation with red or blue light was also abolished by a concomitant far-red light. In leaf pieces of P. sativum blue light was more effective than red, but not in isolated guard cells. In the presence of mesophyll, DCMU inhibited stomatal opening in red light more than in blue, and thus increased the relative response to blue light. This was less evident in isolated guard cells.     

RPT2 is a signal transducer involved in phototropic response and stomatal opening by association with phototropin 1 in Arabidopsis thaliana

Phototropin 1 (phot1) and phot2, which are blue light receptor kinases, function in blue light-induced hypocotyl phototropism, chloroplast relocation, and stomatal opening in Arabidopsis (Arabidopsis thaliana). Previous studies have shown that the proteins RPT2 (for ROOT PHOTOTROPISM2) and NPH3 (for NONPHOTOTROPIC HYPOCOTYL3) transduce signals downstream of phototropins to induce the phototropic response. However, the involvement of RPT2 and NPH3 in stomatal opening and in chloroplast relocation mediated by phot1 and phot2 was unknown. Genetic analysis of the rpt2 mutant and of a series of double mutants indicates that RPT2 is involved in the phot1-induced phototropic response and stomatal opening but not in chloroplast relocation or phot2-induced movements. Biochemical analyses indicate that RPT2 is purified in the crude microsomal fraction, as well as phot1 and NPH3, and that RPT2 makes a complex with phot1 in vivo. On the other hand, NPH3 is not necessary for stomatal opening or chloroplast relocation. Thus, these results suggest that phot1 and phot2 choose different signal transducers to induce three responses: phototropic response of hypocotyl, stomatal opening, and chloroplast relocation.     

The role of a 14-3-3 protein in stomatal opening mediated by PHOT2 in Arabidopsis

The 14-3-3 λ isoform is required for normal stomatal opening mediated by PHOT2 in Arabidopsis thaliana. Arabidopsis phototropin2 (PHOT2) interacts with the λ-isoform 14-3-3 protein both in yeast two-hybrid screening and in an in vitro pull-down assay. Further yeast two-hybrid analysis also showed that the PHOT2 C-terminal kinase domain was required for the interaction. Site-directed mutagenesis indicated that PHOT2 Ser-747 is essential for the yeast interaction. Phenotypic characterization of a loss-of-function 14-3-3 λ mutant in a phot1 mutant background showed that the 14-3-3 λ protein was necessary for normal PHOT2-mediated blue light-induced stomatal opening. PHOT2 Ser-747 was necessary for complementation of the blue light-activated stomatal response in a phot1 phot2 double mutant. The 14-3-3 λ mutant in the phot1 mutant background allowed normal phototropism and normal chloroplast accumulation and avoidance responses. It also showed normal stomatal opening mediated by PHOT1 in a phot2 mutant background. The 14-3-3 κ mutant had no effect on stomatal opening in response to blue light. Although the 14-3-3 λ mutant had no chloroplast movement phenotype, the 14-3-3 κ mutation caused a weaker avoidance response at an intermediate blue light intensity by altering the balance between the avoidance and accumulation responses. The results highlight the strict specificity of phototropin-mediated signal transduction pathways.     

FLOWERING LOCUS T regulates stomatal opening

Stomatal pores surrounded by a pair of guard cells in the plant epidermis control gas exchange for photosynthesis in response to light, CO(2), and phytohormone abscisic acid. Phototropins (phot1 and phot2) are plant blue-light receptor kinases and mediate stomatal opening via activation of the plasma membrane H(+)-ATPase. However, the signaling mechanism from phototropins to the H(+)-ATPase has yet to be determined. Here, we show that FLOWERING LOCUS T (FT) is expressed in guard cells and regulates stomatal opening. We isolated an scs (suppressor of closed-stomata phenotype in phot1 phot2) 1-1 mutant of Arabidopsis thaliana and showed that scs1-1 carries a novel null early flowering 3 (elf3) allele in a phot1 phot2 background. scs1-1 (elf3 phot1 phot2 triple mutant) had an open-stomata phenotype with high H(+)-ATPase activity and showed increased levels of FT mRNA in guard cells. Transgenic plants overexpressing FT in guard cells showed open stomata, whereas a loss-of-function FT allele, ft-1, exhibited closed stomata and failed to activate the H(+)-ATPase in response to blue light. Our results define a new cell-autonomous role for FT and demonstrate that the flowering time genes ELF3 and FT are involved in the regulation of H(+)-ATPase by blue light in guard cells.     

The Arabidopsis rcn1-1 Mutation Impairs Dephosphorylation of Phot2, Resulting in Enhanced Blue Light Responses

Phototropins (phot) sense blue light through the two N-terminal chromophore binding LOV domains and activate the C-terminal kinase domain. The resulting phototropin autophosphorylation is essential for biological activity. We identified the A1 subunit of Ser/Thr protein phosphatase 2A (PP2A) as interacting with full-length phot2 in yeast and also interacting with phot2 in an in vitro protein binding assay. Phenotypic characterizations of a phot1-5 rcn1-1 (for root curling in n-naphthylphthalamic acid1) double mutant, in which phot2 is the only functional phototropin and PP2A activity is reduced, showed enhanced phototropic sensitivity and enhanced blue light–induced stomatal opening, suggesting that PP2A activity is involved in regulating phot2 function. When treated with cantharidin, a chemical inhibitor of PP2A, the phot1-5 mutant exhibited enhanced phot2-mediated phototropic responses like those of the phot1-5 rcn1-1 double mutant. Immunoblot analysis to examine phot2 endogenous phosphorylation levels and in vitro phosphorylation assays of phot2 extracted from plants during dark recovery from blue light exposure confirmed that phot2 is more slowly dephosphorylated in the reduced PP2A activity background than in the wild-type PP2A background, suggesting that phosphorylated phot2 is a substrate of PP2A activity. While reduced PP2A activity enhanced the activity of phot2, it did not enhance either phot1 dephosphorylation or the activity of phot1 in mediating phototropism or stomatal opening.     

Cryptochromes,Phytochromes, and COP1 Regulate Light-Controlled Stomatal Development in Arabidopsis

In Arabidopsis thaliana, the cryptochrome (CRY) blue light photoreceptors and the phytochrome (phy) red/far-red light photoreceptors mediate a variety of light responses. COP1, a RING motif–containing E3 ubiquitin ligase, acts as a key repressor of photomorphogenesis. Production of stomata, which mediate gas and water vapor exchange between plants and their environment, is regulated by light and involves phyB and COP1. Here, we show that, in the loss-of-function mutants of CRY and phyB, stomatal development is inhibited under blue and red light, respectively. In the loss-of-function mutant of phyA, stomata are barely developed under far-red light. Strikingly, in the loss-of-function mutant of either COP1 or YDA, a mitogen-activated protein kinase kinase kinase, mature stomata are developed constitutively and produced in clusters in both light and darkness. CRY, phyA, and phyB act additively to promote stomatal development. COP1 acts genetically downstream of CRY, phyA, and phyB and in parallel with the leucine-rich repeat receptor-like protein TOO MANY MOUTHS but upstream of YDA and the three basic helix-loop-helix proteins SPEECHLESS, MUTE, and FAMA, respectively. These findings suggest that light-controlled stomatal development is likely mediated through a crosstalk between the cryptochrome-phytochrome-COP1 signaling system and the mitogen-activated protein kinase signaling pathway.     

Sugar and Organic Acid Accumulation in Guard Cells of Vicia faba in Response to Red and Blue Light

Changes in neutral sugar and organic acid content of guard cells were quantitated by high-performance liquid chromatography during stomatal opening in different light qualities. Sonicated Vicia faba epidermal peels were irradiated with 10 [mu]mol m-2 s-1 of blue light, a fluence rate insufficient for the activation of guard cell photosynthesis, or 125 [mu]mol m-2 s-1 of red light, in the presence of 1 mM KCl, 0.1 mM CaCl2. The low-fluence-rate blue light stimulated an average net stomatal opening of 4.7 [mu]m in 2 h, whereas the saturating fluence rate of red light stimulated an average net opening of 3.8 [mu]m in 2 h. Under blue light, the malate content of guard cells increased to 173% of the initial level during the first 30 min of opening and declined as opening continued. Sucrose levels continuously rose throughout the blue light-stimulated opening, reaching 215% of the initial level after 2 h. The starch hydrolysis products maltose and maltotriose remained elevated at all times. Under red light, guard cells showed very little increase in organic acid or maltose levels, whereas sucrose levels increased to 208% of the initial level after 2 h. Total measured organic metabolite concentrations were correlated with stomatal apertures in all cases except where substantial malate increases occurred. These results support the hypothesis that light quality modulates alternative mechanisms of osmotic accumulation in guard cells, including potassium uptake, photosynthetic sugar production, and starch breakdown.     

Further support for the involvement of phytoehrome in stomatal movement

The involvement of phytochrome in stomatal movement in Commelina communis L. is indicated by the following observations: 1) Short irradiation with red or blue light causes opening, of isolated stomata and swelling of guard cell protoplasts. This is reversed by subsequent far red irradiation. 2) In a similar way, stomatal response to prolonged irradiation with red or blue light is decreased by concomitant far red irradiation. 3) Pretreatment with filipin, which interferes with phytochrome binding to membranes, decreases stomatal opening in red and blue light. The stomatal responses to blue and red light are modified by DCMU, N₂, CO_2-enriched atmosphere, and CO_2-free air, which are known to affect, among other processes, chlorophyll fluorescence. Increased chlorophyll fluorescence by DCMU, N₂ and CO₂-enriched atmosphere enhanced stomatal opening in blue light and inhibited it in red light. CO₂-free air, which decreases chlorophyll fluorescence, had the opposite effect.     

The blue light-specific response of Vicia faba stomata acclimates to growth environment

Stomata in epidermal strips from growth chamber-grown Vicia faba leaves opened less in response to white light than did stomata from greenhouse-grown leaves. Chlorophyll-mediated, red light-stimulated opening was similar in stomata from the two growth conditions, but stomata from the growth chamber environment had a severely reduced response to blue light. Transfer of plants between the two growth conditions resulted in an acclimation of the stomatal blue light response. Stomata lost blue light sensitivity within 1 d of transfer to growth chamber conditions and gained sensitivity to blue light over an 8 d period after transfer to a greenhouse. Short-term transfer experiments confirmed that the rapid loss of blue light sensitivity was an acclimation response, requiring between 12 and 20 h exposure to growth chamber conditions. The acclimation of the stomatal response to blue light was inversely related to a previously reported acclimation response in which stomata change between high CO2 sensitivity under growth chamber conditions and low CO2 sensitivity under greenhouse conditions. The time courses of the blue light and CO2 acclimation responses were virtually identical, suggesting the possibility of a common acclimation mechanism.     

Functional analyses of the activation loop of phototropin2 in Arabidopsis

Phototropins (phot1 and phot2) are autophosphorylating blue-light receptor kinases that mediate blue-light responses such as phototropism, chloroplast accumulation, and stomatal opening in Arabidopsis (Arabidopsis thaliana). Only phot2 induces the chloroplast avoidance response under strong blue light. The serine (Ser) residues of the kinase activation loop in phot1 are autophosphorylated by blue light, and autophosphorylation is essential for the phot1-mediated responses. However, the role of autophosphorylation in phot2 remains to be determined. In this study, we substituted the conserved residues of Ser-761 and Ser-763 with alanine (S761A S763A) in the phot2 activation loop and analyzed their function by investigating the phot2-mediated responses after the transformation of phot1 phot2 double mutant with this mutant phot2 gene. Transgenic plants expressing the mutant phot2 protein exhibited impaired responses in chloroplast movement, stomatal opening, phototropic bending, leaf flattening, and plant growth; and those expressing phot2 with S761D S763D mutations showed the normal responses. Substitution of both Ser-761 and Ser-763 with alanine in phot2 did not significantly affect the kinase activity in planta. From these results, we conclude that phosphorylation of Ser-761 and Ser-763 in the activation loop may be a common primary step for phot2-mediated responses.