Intramolecular co-action of two independent photosensory modules in the fern phytochrome 3

Fern phytochrome3/neochrome1 (phy3/neo1) is a chimeric photoreceptor composed of a phytochrome-chromophore binding domain and an almost full-length phototropin. phy3 thus contains two different light-sensing modules; a red/far-red light receptor phytochrome and a blue light receptor phototropin. phy3 induces both red light- and blue light-dependent phototropism in phototropin-deficient Arabidopsis thaliana (phot1 phot2) seedlings. The red-light response is dependent on the phytochrome module of phy3, and the blue-light response is dependent on the phototropin module. We recently showed that both the phototropin-sensing module and the phytochrome-sensing module mediate the blue light-dependent phototropic response. Particularly under low-light conditions, these two light-sensing modules cooperate to induce the blue light-dependent phototropic response. This intramolecular co-action of two independent light-sensing modules in phy3 enhances light sensitivity, and perhaps allowed ferns to adapt to the low-light canopy conditions present in angiosperm forests.     

The phototropin family as photoreceptors for blue light-induced chloroplast relocation

Blue light-induced chloroplast accumulation and avoidance relocation movements are controlled by the blue light photoreceptor phototropin. The Arabidopsis thaliana genome has two phototropin genes encoding phot1 and phot2. Each of these photoreceptors contains two LOV (light oxygen and voltage) domains and a kinase domain. The LOV domains absorb blue light though an associated flavin mononucleotide chromophore, while the kinase domain is thought to be associated with signal transduction. The phototropins control not only chloroplast relocation movement, but also blue light-induced phototropic responses, leaf expansion and stomatal opening. Here I review the role of phototropin as a photoreceptor for chloroplast photorelocation movement. Electronic Publication     

Phototropin LOV domains exhibit distinct roles in regulating photoreceptor function

Phototropins (phot1 and phot2) are autophosphorylating serine/threonine kinases that function as photoreceptors for phototropism, light-induced chloroplast movement, and stomatal opening in Arabidopsis. The N-terminal region of phot1 and phot2 contains two specialized PAS domains, designated LOV1 and LOV2, which function as binding sites for the chromophore flavin mononucleotide (FMN). Both LOV1 and LOV2 undergo a self-contained photocycle, which involves the formation of a covalent adduct between the FMN chromophore and a conserved active-site cysteine residue (Cys39). Replacement of Cys39 with alanine abolishes the light-induced photochemical reaction of LOV1 and LOV2. Here we have used the Cys39Ala mutation to investigate the role of LOV1 and LOV2 in regulating phototropin function. Photochemical analysis of a bacterially expressed LOV1 + LOV2 fusion protein indicates that LOV2 functions as the predominant light-sensing domain for phot1. LOV2 also plays a major role in mediating light-dependent autophosphorylation of full-length phot1 expressed in insect cells and transgenic Arabidopsis. Moreover, photochemically active LOV2 alone in full-length phot1 is sufficient to elicit hypocotyl phototropism in transgenic Arabidopsis, whereas photochemically active LOV1 alone is not. Further photochemical and biochemical analyses also indicate that the LOV1 and LOV2 domains of phot2 exhibit distinct roles. The significance for the different roles of the phototropin LOV domains is discussed.     

Stability of dimer and domain-domain interaction of Arabidopsis phototropin 1 LOV2

Transient grating signals after photoexcitation of Arabidopsis phototropin 1 light-oxygen-voltage 2 (phot1LOV2) domain without the linker were found to be very sensitive to temperature. In particular, the diffusion signal drastically increased with rising temperature. The signal was consistently explained by the superposition of the photo-induced dissociation and association reactions. This observation indicated the presence of an equilibrium between the monomer and dimer forms of the phot1LOV2 domain in the dark. The equilibrium was confirmed by a gel chromatographic technique. The equilibrium constants at various temperatures were calculated from the fraction of the dimer, and the stabilization enthalpy and entropy were determined. Interestingly, the transient grating signal of phot1LOV2 with the linker (phot1LOV2-linker), which exists as the monomer form, was also temperature dependent; the diffusion signal intensity decreased with increasing temperature. Because the diffusion signal reflects a conformation change of the linker upon photoexcitation, this temperature dependence indicated that there were two forms of the phot1LOV2-linker. One form exhibited a conformational change upon photoexcitation whereas the other form showed no change. These two forms are not distinguishable spectroscopically. The fraction of these species depended on the temperature. Considering the monomer-dimer equilibrium of the phot1LOV2 domain, we suggest that the nonreactive form possesses the linker region that is dissociated from the LOV2 domain. Because the dissociation of the linker region from the LOV2 domain is a key step for the conformation change of the phot1LOV2-linker to induce biological activity, we proposed that the phototropins could have a role as a temperature sensor.     

Oligomeric structure of LOV domains in Arabidopsis phototropin

Oligomeric structures of the four LOV domains in Arabidopsis phototropin1 (phot1) and 2 (phot2) were studied using crosslinking. Both LOV1 domains of phot1 and phot2 form a dimer independently on the light conditions, suggesting that the LOV1 domain can be a stable dimerization site of phot in vivo. In contrast, phot1-LOV2 is in a monomer-dimer equilibrium and phot2-LOV2 exists as a monomer in the dark. Blue light-induced a slight increase in the monomer population in phot1-LOV2, suggesting a possible blue light-inducible dissociation of dimers. Furthermore, blue light caused a band shift of the phot2-LOV2 monomer. CD spectra revealed the unfolding of helices and the formation of strand structures. Both light-induced changes were reversible in the dark.

Structured summary

MINT-6823377, MINT-6823391:PHOT1 (uniprotkb:O48963) and PHOT1 (uniprotkb: O48963) bind (MI:0407) by cross-linking studies (MI:0030)MINT-6823495, MINT-6823508:PHOT2 (uniprotkb:P93025) and PHOT2 (uniprotkb:P93025) bind (MI:0407) by cross-linking studies (MI:0030)     

Phototropins and Their LOV Domains：Versatile Plant Blue-Light Receptors

The phototropins phot1 and phot2 are plant blue-light receptors that mediate phototropism, chloroplast movements, stomatal opening, leaf expansion, the rapid Inhibition of hypocotyl growth in etiolated seedlings, and possibly solar tracking by leaves in those species in which It occurs. The phototroplns are plasma membrane-associated hydrophilic proteins with two chromophore domains （designated LOV1 and LOV2 for their resemblance to domains In other signaling proteins that detect light, oxygen, or voltage） in their Nterminal half and a classic serine/threonlne kinase domain in their C-terminal half. Both chromophore domains bind flavin mononucleotide （FMN） and both undergo light-activated formation of a covalent bond between a nearby cystelne and the C（4a） carbon of the FMN to form the signaling state. LOV2-cystelnyl adduct formation leads to the release downstream of a tightly bound amphlpathlc α-helix, a step required for activation of the klnase function. This cysteinyl adduct then slowly decays over a matter of seconds or minutes to return the photoreceptor chromophore modules to their ground state. Functional LOV2 is required for light-activated phosphorylation and for various blue-light responses mediated by the phototroplns. The function of LOV1 is still unknown, although It may serve to modulate the signal generated by LOV2. The LOV domain Is an ancient chromophore module found In a wide range of otherwise unrelated proteins In fungi and prokaryotes, the latter Including cyanobacterla, eubacterla, and archaea. Further general reviews on the phototropins are those by Celaya and Liscum （2005） and Christie and Briggs （2005）.     

Photochemical intermediates of Arabidopsis phototropin 2 LOV domains associated with conformational changes

The photochemical reactions of Arabidopsis phototropin 2 light- oxygen-voltage domain 2 (LOV2) with the linker region (LOV2-linker), without the linker (LOV2), and LOV1 were studied using the time-resolved transient grating (TG) and transient lens (TrL) methods. Although the absorption spectra did not change after the formation of the adduct species, a small volume expansion process with a time constant of 9 ms was observed for LOV2. For the LOV2-linker, at 293 K, a volume contraction process with a time constant of 140 mus was observed in addition to a volume expansion process with 9 ms and the diffusion coefficient change with 2 ms. The reaction intermediate species were characterized on the basis of their thermodynamic properties, such as changes in enthalpy, thermal expansion, and heat capacity. For the first intermediate (S(390)), the values of these properties were similar to those of the ground state for both LOV2 and LOV2-linker. A relatively large thermal expansion volume (0.09 cm(3)mol(-1)K(-1)) and a positive heat capacity change (4.7 kJ mol(-1)K(-1)) were detected for the intermediates of LOV2-linker. These characteristic features were interpreted in terms of structural fluctuation and exposure of hydrophobic residues in the linker domain, respectively. The enthalpy change of S(390) of the LOV1 domain was significantly greater than changes for the LOV2 or LOV2-linker samples. Data from this study support a major conformational change of the linker region in the photochemical reaction of phototropin.     

Structural basis of the LOV1 dimerization of Arabidopsis phototropins 1 and 2

Phototropin (phot) is a blue-light receptor protein that triggers phototropic responses, chloroplast relocation, and stomata opening to maximize the efficiency of photosynthesis in higher plants. Phot is composed of three functional domains. The N-terminal half folds into two light-oxygen-voltage-sensing domains called LOV1 and LOV2, each binding a flavin mononucleotide to absorb blue light. The C-terminal half is a serine/threonine kinase domain that causes light-dependent autophosphorylation leading to cellular signaling cascades. LOV2 domain is primarily responsible for activation of the kinase, and LOV1 domain is thought to act as a dimerization site and to regulate sensitivity to activation by blue light. Here we show the crystal structures of LOV1 domains of Arabidopsis phot1 and phot2 in the dark at resolutions of 2.1 Å and 2.0 Å, respectively. Either LOV1 domain forms a dimer through face-to-face association of β-scaffolds in the crystallographic asymmetric unit. Three types of interactions stabilizing the dimer structures found are as follows: contacts of side chains in their β-scaffolds, hydrophobic interactions of a short helix found in the N-terminus of a subunit with the β-scaffolds of both subunits, and hydrogen bonds mediated by hydration water molecules filling the dimer interface. The critical residues for dimerization are Cys261, forming a disulfide bridge between subunits in phot1-LOV1 domain, and Thr217 and Met232 in phot2-LOV1. The topology in homodimeric associations of the LOV1 domains is discussed when referring to those of homodimers or heterodimers of light-oxygen-voltage-sensing or Per-ARNT-Sim domains. The present results also provide clues to understanding structural basis in dimeric interactions of Per-ARNT-Sim protein modules in cellular signaling.     

Phytochrome regulation of pea phototropin

Type 1 phototropin, one of the blue light receptors responsible for phototropism, is encoded in peas by at least two genes, PsPHOT1A and PsPHOT1B (formerly PsPK4 and PsPK5), both of which are more similar to Arabidopsis PHOT1 than to Arabidopsis PHOT2. We show here that PsPHOT1B encodes a full-length phototropin, whose expression pattern suggests that Psphot1b is the predominant phot1-type phototropin in etiolated seedlings. The gene encoding the other type 1 phototropin, PsPHOT1A, is expressed at low levels, with its highest levels in the leaves and stems of more mature, light-grown plants. Studies with phyA, phyB and the phyAphyB double mutants show that phyA and phyB have partially redundant roles in the reduction of PsPHOT1B expression under red light.     

The LOV domain: a chromophore module servicing multiple photoreceptors

Three different families of blue-light receptors have been characterized from higher plants: three cryptochromes, two phototropins, and the three members of the ZTL/ADO family. Phototropins and the ZTL/ADO proteins have chromophore modules, designated LOV domains, that bind flavin mononucleotide and undergo formation of a C(4a) flavin-cysteinyl adduct. All contain the highly conserved amino acid motif GXNCRFLQ. Over 90 prokaryote proteins also contain LOV domains with this motif upstream from one of several different functional groups. All of these that have been investigated to date act as photoreceptors in vitro and form the adduct upon irradiation. Four members of the class LOV-histidine kinase, one from a plant pathogen (Pseudomonas syringae), one from an animal pathogen Brucella melitensis), and two from a marine bacterium (Erythrobacter litoralis) respectively, mediate light-activated histidine phosphorylation. Decay of the adduct in darkness after a blue light pulse coincides with loss of the capacity for phosphorylation upon addition of ATP. At present, the biological role(s) of these light-sensitive proteins is under investigation.     

Photochemical and mutational analysis of the FMN-binding domains of the plant blue light receptor, phototropin

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.     

A conserved isoleucine in the LOV1 domain of a novel phototropin from the marine alga Ostreococcus tauri modulates the dark state recovery of the domain

Background

Phototropins are UV-A/blue light receptor proteins with two LOV (Light-Oxygen-Voltage) sensor domains at their N terminus and a kinase domain at the C-terminus in photoautotrophic organisms. This is the first research report of a canonical phototropin from marine algae Ostreococcus tauri.

Methods

We synthesized core LOV1 (OtLOV1) domain-encoding portion of the phototropin gene of O. tauri, the domain was heterologously expressed, purified and assessed for its spectral properties and dark recovery kinetics by UV–Visible, fluorescence spectroscopy and mutational studies. Quaternary structure characteristics were studied by SEC and glutaraldehyde crosslinking.

Results

The absorption spectrum of OtLOV1 lacks the characteristic 361 nm peak shown by other LOV1 domains. It undergoes a photocycle with a dark state recovery time of approximately 30 min (τ = 300.35 s). Native OtLOV1 stayed as dimer in aqueous solution and the dimer formation was light and concentration independent. Mutating isoleucine at 43rd position to valine accelerated the dark recovery time by more than 10-fold. Mutating it to serine reduced sensitivity to blue light, but the dark recovery time remained unaltered. I43S mutation also destabilized the FMN binding to a great extent.

Conclusion

The OtLOV1 domain of the newly identified OtPhot is functional and the isoleucine at position 43 of OtLOV1 is the key residue responsible for fine-tuning the domain properties.

General significance

This is the first characterized LOV1 domain of a canonical phototropin from a marine alga and spectral properties of the domain are similar to that of the LOV1 domain of higher plants.     

Mapping of low- and high-fluence autophosphorylation sites in phototropin 1

Phototropins, originally detected by their blue light-dependent autophosphorylation, are plant photoreceptors involved in several blue light responses such as phototropism, chloroplast relocation, leaf expansion, rapid inhibition of hypocotyl growth, and stomatal opening. Three domains have been identified in phototropin sequences, two chromophore binding domains (LOV1 and LOV2) and a kinase domain. We describe here two additional domains, the N-terminus upstream of LOV1 and the hinge region between LOV1 and LOV2, as the regions for autophosphorylation; the phosphorylation sites were identified by site-directed mutagenesis as S27, S30, S274, S300, S317, S325, S332, and S349 of the PHOT1a sequence of Avena sativa. Investigation of the autophosphorylation in vivo revealed that serines close to the LOV1 domain are phosphorylated at lower fluence of blue light than the serines close to the LOV2 domain. Recovery of phosphorylation in vivo during a dark period after saturating irradiation is caused by dephosphorylation rather than by degradation of the phosphorylated form and new synthesis of nonphosphorylated phototropin. The results were obtained by a combination of autophosphorylation of phototropin with phosphorylation of recombinant domains by protein kinase A, which turned out to have the same site specificity as the phototropin kinase, followed by proteolysis and separation of phosphopeptides. With the knowledge of the phosphorylation sites, the physiological and biochemical consequences of autophosphorylation can now be approached by site-directed mutagenesis of phototropins.     

LOVe across kingdoms: Blue light perception vital for growth and development in plant–fungal interactions

Blue light plays an important role in the growth and development of fungi. Environmental cues enable plant pathogenic fungi to synchronise essential metabolic pathways to that of their hosts to gain a competitive advantage. Phylogenetic analysis of the LOV domain present in blue light receptors across all three kingdoms suggests that these receptors in fungal lineages have undergone convergent evolution to use the same domain for control and regulation of similar cellular and metabolic processes. In this review, the genetic basis of blue light photoperception in fungi, and the functions it regulates, will be discussed. Furthermore, the evolution of the light sensing domain and its role in pathogenesis is hypothesised concluding with how knowledge of conserved LOV domains may be exploited for fungal disease control in crop plants.     

Molecular basis of the functional specificities of phototropin 1 and 2

A blue-light photoreceptor in plants, phototropin, mediates phototropism, chloroplast relocation, stomatal opening, and leaf-flattening responses. Phototropin is divided into two functional moieties, the N-terminal photosensory and the C-terminal signaling moieties. Phototropin perceives light stimuli by the light, oxygen or voltage (LOV) domain in the N-terminus; the signal is then transduced intramolecularly to the C-terminal kinase domain. Two phototropins, phot1 and phot2, which have overlapping and distinct functions, exist in Arabidopsis thaliana. Phot1 mediates responses with higher sensitivity than phot2. Phot2 mediates specific responses, such as the chloroplast avoidance response and chloroplast dark positioning. To elucidate the molecular basis for the functional specificities of phot1 and phot2, we exchanged the N- and C-terminal moieties of phot1 and phot2, fused them to GFP and expressed them under the PHOT2 promoter in the phot1 phot2 mutant background. With respect to phototropism and other responses, the chimeric phototropin consisting of phot1 N-terminal and phot2 C-terminal moieties (P1n/2cG) was almost as sensitive as phot1; whereas the reverse combination (P2n/1cG) functioned with lower sensitivity. Hence, the N-terminal moiety mainly determined the sensitivity of the phototropins. Unexpectedly, both P1n/2cG and P2n/1cG mediated the chloroplast avoidance response, which is specific to phot2. Hence, chloroplast avoidance activity appeared to be suppressed specifically in the combination of N- and C-terminal moieties of phot1. Unlike the chloroplast avoidance response, chloroplast dark positioning was observed for P2G and P2n/1cG but not for P1G or P1n/2cG, suggesting that a specific structure in the N-terminal moiety of phot2 is required for this activity.     

Recent advances in understanding the molecular mechanism of chloroplast photorelocation movement

Plants are photosynthetic organisms that have evolved unique systems to adapt fluctuating environmental light conditions. In addition to well-known movement responses such as phototropism, stomatal opening, and nastic leaf movements, chloroplast photorelocation movement is one of the essential cellular responses to optimize photosynthetic ability and avoid photodamage. For these adaptations, chloroplasts accumulate at the areas of cells illuminated with low light (called accumulation response), while they scatter from the area illuminated with strong light (called avoidance response). Plant-specific photoreceptors (phototropin, phytochrome, and/or neochrome) mediate these dynamic directional movements in response to incident light position and intensity. Several factors involved in the mechanisms underlying the processes from light perception to actin-based movements have also been identified through molecular genetic approach. This review aims to discuss recent findings in the field relating to how chloroplasts move at molecular levels. This article is part of a Special Issue entitled: Dynamic and ultrastructure of bioenergetic membranes and their components.     

Vibrational spectroscopy of an algal Phot-LOV1 domain probes the molecular changes associated with blue-light reception

The LOV1 domain of the blue light Phot1-receptor (phototropin homolog) from Chlamydomonas reinhardtii has been studied by vibrational spectroscopy. The FMN modes of the dark state of LOV1 were identified by preresonance Raman spectroscopy and assigned to molecular vibrations. By comparing the blue-light-induced FTIR difference spectrum with the preresonance Raman spectrum, most of the differences are due to FMN modes. Thus, we exclude large backbone changes of the protein that might occur during the phototransformation of the dark state LOV1-447 into the putative signaling state LOV1-390. Still, the presence of smaller amide difference bands cannot be excluded but may be masked by overlapping FMN modes. The band at 2567 cm(-1) is assigned to the S-H stretching vibration of C57, the residue that forms the transient thio-adduct with the chromophore FMN. The occurrence of this band is evidence that C57 is protonated in the dark state of LOV1. This result challenges conclusions from the homologous LOV2 domain from oat that the thiolate of the corresponding cysteine is the reactive species.     

Structural basis for light-dependent signaling in the dimeric LOV domain of the photosensor YtvA

The photosensor YtvA binds flavin mononucleotide and regulates the general stress reaction in Bacillus subtilis in response to blue light illumination. It belongs to the family of light-oxygen-voltage (LOV) proteins that were first described in plant phototropins and form a subgroup of the Per-Arnt-Sim (PAS) superfamily. Here, we report the three-dimensional structure of the LOV domain of YtvA in its dark and light states. The protein assumes the global fold common to all PAS domains and dimerizes via a hydrophobic interface. Directly C-terminal to the core of the LOV domain, an alpha-helix extends into the solvent. Light absorption causes formation of a covalent bond between a conserved cysteine residue and atom C(4a) of the FMN ring, which triggers rearrangements throughout the LOV domain. Concomitantly, in the dark and light structures, the two subunits of the dimeric protein rotate relative to each other by 5 degrees . This small quaternary structural change is presumably a component of the mechanism by which the activity of YtvA is regulated in response to light. In terms of both structure and signaling mechanism, YtvA differs from plant phototropins and more closely resembles prokaryotic heme-binding PAS domains.     

Quaternary structure of LOV-domain containing polypeptide of Arabidopsis FKF1 protein

Flavin-binding, Kelch repeat, F-box (FKF1) protein is a photoreceptor to regulate flowering of Arabidopsis. The protein has a light, oxygen and voltage (LOV)-sensing domain binding a flavin mononucleotide. The photo-activation of the domain is an indispensable step to initiate the cellular signaling for flowering. In the present study, a LOV-containing polypeptide of FKF1 was prepared by an overexpression system, and the quaternary structure of it was studied by size exclusion chromatography and small-angle X-ray scattering. The apparent molecular weight from chromatography suggested a globular trimeric or an anisotropic-shaped dimeric association of the polypeptide in solution. The scattering experiment demonstrated a dimeric association of the polypeptides with an elongated molecular shape displaying the radius of gyration of 27 A and the maximum dimension of 94 A. The molecular shape simulated from scattering profiles suggests an antiparallel association of the LOV domains in the dimer. Though the absorption spectrum of blue-light irradiated polypeptide was stable in the photoactivated state for a long period, the scattering profiles showed very small changes between the dark and light conditions. Based on the homologies in the amino-acid sequences and the scattering profiles, these results are discussed in connection with the structures and function of LOV domains of phototropin.     

13C Isotopologue editing of FMN bound to phototropin domains

The plant blue light receptor phototropin comprises a protein kinase domain and two FMN-binding LOV domains (LOV1 and LOV2). Blue light irradiation of recombinant LOV domains is conducive to the addition of a cysteinyl thiolate group to carbon 4a of the FMN chromophore, and spontaneous cleavage of that photoadduct completes the photocycle of the receptor. The present study is based on (13)C NMR signal modulation observed after reconstitution of LOV domains of different origins with random libraries of (13)C-labeled FMN isotopologues. Using this approach, all (13)C signals of FMN bound to LOV1 and LOV2 domains of Avena sativa and to the LOV2 domain of the fern, Adiantum capillus-veneris, could be unequivocally assigned under dark and under blue light irradiation conditions. (13)C Chemical shifts of FMN are shown to be differently modulated by complexation with the LOV domains under study, indicating slight differences in the binding interactions of FMN and the apoproteins.