The plant blue light receptor, phot1, a member of the phototropin family (1), is a plasma membrane-associated flavoprotein that contains two (ϳ110 amino acids) flavinbinding domains, LOV1 and LOV2, within its N terminus and a typical serine-threonine protein kinase domain at its C terminus. The LOV (light, oxygen, and voltage) domains belong to the PAS domain superfamily of sensor proteins. In response to blue light, phototropins undergo autophosphorylation. E. coli-expressed LOV domains bind riboflavin-5-monophosphate, are photochemically active, and have major absorption peaks at 360 and 450 nm, with the 450 nm peak having vibronic structure at 425 and 475 nm. These spectral features correspond to the action spectrum for phototropism in higher plants. Near-UV blue light regulates a variety of different responses in higher plants. These include phototropism, the inhibition of hypocotyl elongation, the expression of various genes, and stomatal opening. Phot1 (nph1), the recently discovered blue light receptor, is a member of the phototropin receptor family (1). Phot1 is a plasma membrane-associated flavoprotein that functions as the primary photoreceptor mediating phototropic plant movement (2-4). Phot1 has two 12.1-kDa flavin-binding domains, LOV1 and LOV2, within its N-terminal region and a typical serinethreonine protein kinase domain at the C-terminal region. Heterologous expression studies have shown that phot1 binds FMN 1 as a chromophore and undergoes autophosphorylation in response to light treatment. It has therefore been proposed that this receptor functions as a light-activated serine/threonine kinase (4). The isolated LOV domains from oat phot1 expressed in Escherichia coli have been shown to undergo a cyclic photoreaction upon the absorption of light; LOV1 recovers with a half-time of 11.5 s, whereas LOV2 recovers with a half-time of 27 s (5). In addition, the quantum efficiencies for photoproduct (adduct) formation for LOV1 and LOV2 are ϳ0.045 and 0.44, respectively (5). The ground forms of the LOV domains have major absorption peaks at 360 and 450 nm with the 450 peak having vibronic structure at 425 and 475 nm. Upon absorption of light, the chromophore bleaches 2 in the 450 nm region generating a species that absorbs maximally at 390 nm. This intermediate has been assigned as a flavin-cysteinyl adduct between the protein and the C(4a) carbon of the FMN chromophore. This adduct breaks down spontaneously, returning the protein to its ground form. A LOV2 mutant (LOV2C39A) in which the cysteine that forms the adduct has been mutated to alanine does not undergo this photoreaction (5).Recently the crystal structure of the LOV2 domain from the fern Adiantum capillus-veneris phy3 (6) was solved to 2.7-Å resolution (7). Phy3 is a chimeric photoreceptor with homology to phytochrome at its N-terminal end and an almost complete phototropin at its C-terminal end. Its LOV2 domain shares a 70% sequence homology to the oat phot1 LOV2 (6). The structure indicates that the FMN molecule is held noncovalently within...

The plant photoreceptor phototropin is an autophosphorylating serine-threonine protein kinase activated by UV-A/blue light. Two domains, LOV1 and LOV2, members of the PAS domain superfamily, mediate light sensing by phototropin. Heterologous expression studies have shown that both domains function as FMN-binding sites. Although three plant blue light photoreceptors, cry1, cry2, and phototropin, have been identified to date, the photochemical reactions underlying photoactivation of these light sensors have not been described so far. Herein, we demonstrate that the LOV domains of Avena sativa phototropin undergo a self-contained photocycle characterized by a loss of blue light absorbance in response to light and a spontaneous recovery of the blue light-absorbing form in the dark. Rate constants and quantum efficiencies for the photoreactions indicate that LOV1 exhibits a lower photosensitivity than LOV2. The spectral properties of the photoproduct produced for both LOV domains are unrelated to those found for photoreduced flavins and flavoproteins, but are consistent with those of a flavin-cysteinyl adduct. Flavin-thiol adducts are generally short-lifetime reaction intermediates formed during the flavoprotein-catalyzed reduction of protein disulfides. By site-directed mutagenesis, we have identified several amino acid residues within the putative chromophore binding site of LOV1 and LOV2 that appear to be important for FMN binding and/or the photochemical reactivity. Among those is Cys39, which plays an important role in the photochemical reaction of the LOV domains. Replacement of Cys39 with Ala abolished the photochemical reactions of both LOV domains. We therefore propose that light sensing by the phototropin LOV domains occurs via the formation of a stable adduct between the FMN chromophore and Cys39.

Phototropins are blue-light receptors controlling a range of responses that serve to optimize the photosynthetic efficiency of plants. These include phototropism, light-induced stomatal opening, and chloroplast movements in response to changes in light intensity. Since the isolation of the Arabidopsis PHOT1 gene in 1997, phototropins have been identified in ferns and mosses where their physiological functions appear to be conserved. Arabidopsis contains two phototropins, phot1 and phot2, that exhibit overlapping functions in addition to having unique physiological roles. Phototropins are light-activated serine/threonine protein kinases. Light sensing by the phototropins is mediated by a repeated motif at the N-terminal region of the protein known as the LOV domain. Photoexcitation of the LOV domain results in receptor autophosphorylation and an initiation of phototropin signaling. Here we summarize the photochemical and biochemical events underlying phototropin activation in addition to the current knowledge of the molecular mechanisms associated with photoreceptor signaling.

UV-A͞blue light acts to regulate a number of physiological processes in higher plants. These include light-driven chloroplast movement and phototropism. The NPH1 gene of Arabidopsis encodes an autophosphorylating protein kinase that functions as a photoreceptor for phototropism in response to low-intensity blue light. However, nph1 mutants have been reported to exhibit normal phototropic curvature under high-intensity blue light, indicating the presence of an additional phototropic receptor. A likely candidate is the nph1 homologue, npl1, which has recently been shown to mediate the avoidance response of chloroplasts to high-intensity blue light in Arabidopsis. Here we demonstrate that npl1, like nph1, noncovalently binds the chromophore flavin mononucleotide (FMN) within two specialized PAS domains, termed LOV domains. Furthermore, when expressed in insect cells, npl1, like nph1, undergoes light-dependent autophosphorylation, indicating that npl1 also functions as a light receptor kinase. Consistent with this conclusion, we show that a nph1 npl1 double mutant exhibits an impaired phototropic response under both low-and highintensity blue light. Hence, npl1 functions as a second phototropic receptor under high fluence rate conditions and is, in part, functionally redundant to nph1. We also demonstrate that both chloroplast accumulation in response to low-intensity light and chloroplast avoidance movement in response to high-intensity light are lacking in the nph1 npl1 double mutant. Our findings therefore indicate that nph1 and npl1 show partially overlapping functions in two different responses, phototropism and chloroplast relocation, in a fluence rate-dependent manner.L ight is an important environmental factor controlling plant growth and development. In particular, wavelengths in UV-A (320-390 nm) and blue (390-500 nm) regions of the electromagnetic spectrum act to regulate a range of different plant responses. These processes include de-etiolation, photoentrainment of the circadian clock, floral initiation, phototropic curvature, chloroplast relocation, and stomatal opening (1-3). Much of our understanding of blue light perception in higher plants has come from the isolation of blue-light-response mutants of Arabidopsis thaliana. Indeed, molecular genetic studies have shown that the effects of blue light on plant development are mediated by at least four different blue-light receptors in Arabidopsis: cryptochrome 1 (cry1), cryptochrome 2 (cry2), phototropin (nph1, for non-phototropic hypocotyl 1), and the npl1 (nph1-like 1) protein.The phototropin photoreceptor, nph1, mediates both root and hypocotyl phototropism in response to low-fluence-rate unilateral blue light (Ͻ1 mol⅐m Ϫ2 ⅐s Ϫ1 ) (4, 5). Nph1 is a 120-kDa plasma-membrane-associated protein that contains a serine͞ threonine kinase domain located within its C terminus. Furthermore, the N-terminal region of nph1 contains a repeated motif of 110 aa, designated LOV1 and LOV2, that belong to the PAS domain (found in PER, ARNT, and SIM proteins) superfamil...

The recently identified plant photoreceptor UVR8 triggers regulatory changes in gene expression in response to ultraviolet-B (UV-B) light via an unknown mechanism. Here, crystallographic and solution structures of the UVR8 homodimer, together with mutagenesis and far-UV circular dichroism spectroscopy, reveal its mechanisms for UV-B perception and signal transduction. β-propeller subunits form a remarkable, tryptophan-dominated, dimer interface stitched together by a complex salt-bridge network. Salt-bridging arginines flank the excitonically coupled cross-dimer tryptophan “pyramid” responsible for UV-B sensing. Photoreception reversibly disrupts salt bridges, triggering dimer dissociation and signal initiation. Mutation of a single tryptophan to phenylalanine re-tunes the photoreceptor to detect UV-C wavelengths. Our analyses establish how UVR8 functions as a photoreceptor without a prosthetic chromophore to promote plant development and survival in sunlight.

The NPH1 gene of Arabidopsis thaliana encodes a 120-kilodalton serine-threonine protein kinase hypothesized to function as a photoreceptor for phototropism. When expressed in insect cells, the NPH1 protein is phosphorylated in response to blue light irradiation. The biochemical and photochemical properties of the photosensitive protein reflect those of the native protein in microsomal membranes. Recombinant NPH1 noncovalently binds flavin mononucleotide, a likely chromophore for light-dependent autophosphorylation. The fluorescence excitation spectrum of the recombinant protein is similar to the action spectrum for phototropism, consistent with the conclusion that NPH1 is an autophosphorylating flavoprotein photoreceptor mediating phototropic responses in higher plants.

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Phototropism, the bending response of plant organs to or away from a directional light source, is one of the best studied blue light responses in plants. Although phototropism has been studied for more than a century, recent advances have improved our understanding of the underlying signaling mechanisms involved. The NPH1 gene of Arabidopsis thaliana encodes a blue light-dependent autophosphorylating protein kinase with the properties of a photoreceptor for phototropism. NPH1 apoprotein noncovalently binds FMN to form the holoprotein nph1. The N-terminal region of the protein contains two LOV (light, oxygen, or voltage) domains that share homology with sensor proteins from a diverse group of organisms. These include the bacterial proteins NIFL and AER, both of which bind FAD, and the phy3 photoreceptor from Adiantium capillus-veneris. The LOV domain has therefore been proposed to ref lect a f lavin-binding site, regulating nph1 kinase activity in response to blue light-induced redox changes. Herein we demonstrate that the LOV domains of two nph1 proteins and phy3 bind stoichiometric amounts of FMN when expressed in Escherichia coli. The spectral properties of the chromopeptides are similar to the action spectrum for phototropism, implying that the LOV domain binds FMN to function as a light sensor. Thus, our findings support the earlier model that nph1 is a dual-chromophoric f lavoprotein photoreceptor regulating phototropic responses in higher plants. We therefore propose the name phototropin to designate the nph1 holoprotein.