Effect of circularly polarized light on germination, hypocotyl elongation and biomass production of arabidopsis and lettuce: Involvement of phytochrome B.

Circular dichroism (CD), defined as the differential absorption of left- and right-handed circularly polarized light (CPL), is a useful spectroscopic technique for structural studies of biological systems composed of chiral molecules. The present study evaluated the effects of CPL on germination, hypocotyl elongation and biomass production of Arabidopsis and lettuce. Higher germination rates were observed when Arabidopsis and lettuce seedlings were irradiated with red right-handed CPL (R-CPL) than with red left-handed CPL (L-CPL). Hypocotyl elongation was effectively inhibited when Arabidopsis and lettuce seedlings were irradiated with red R-CPL than with red L-CPL. This difference was not observed when a phytochrome B (phyB) deficient mutant of Arabidopsis was irradiated, suggesting that inhibition of elongation by red R-CPL was mediated by phyB. White R-CPL induced greater biomass production by adult Arabidopsis plants, as determined by their fresh shoot weight, than white L-CPL. To determine the molecular basis of these CPL effects, CD spectra and the effect of CPL on the photoreaction of a sensory module of Arabidopsis phyB were measured. The red light-absorbing form of phyB showed a negative CD in the red light-absorbing region, consistent with the results of germination, inhibition of hypocotyl elongation and biomass production. L-CPL and R-CPL, however, did not differ in their ability to induce the interconversion of the red light-absorbing and far-red light-absorbing forms of phyB. These findings suggest that these CPL effects involve phyB, along with other photoreceptors and the photosynthetic process.

Photoreaction Dynamics of Full-Length Phototropin from Chlamydomonas reinhardtii.

Phototropin (phot) is a blue light sensor involved in the light responses of several species from green algae to higher plants. Phot consists of two photoreceptive domains (LOV1 and LOV2) and a Ser/Thr kinase domain. These domains are connected by a hinge and a linker domain. So far, studies on the photochemical reaction dynamics of phot have been limited to short fragments, and the reactions of intact phot have not been well elucidated. Here, the photoreactions of full-length phot and of several mutants from Chlamydomonas reinhardtii (Cr) were investigated by the transient grating and circular dichroism (CD) methods. Full-length Cr phot is in monomeric form in both dark and light states and shows conformational changes upon photoexcitation. When LOV1 is excited, the hinge helix unfolds with a time constant of 77 ms. Upon excitation of LOV2, the linker helix unfolds initially followed by a tertiary structural change of the kinase domain with a time constant of 91 ms. The quantum yield of conformational change after adduct formation of LOV2 is much smaller than that of LOV1, indicating that reactive and nonreactive forms exist. The conformational changes associated with the excitations of LOV1 and LOV2 occur independently and additively, even when they are excited simultaneously. Hence, the role of LOV1 is not to enhance the kinase activity in addition to LOV2 function; we suggest LOV1 has different functions such as regulation of intermolecular interactions.

Measurements of Photoreaction and Kinase Activity of Phototropin, a Photoreceptor Protein for Tropic Response in Plants: Involvement of Kinase Activity in the Photosensitivity of Tropic Response.

Phototropin is a photoreceptor protein responsible for phototropic responses in plants. A phototropin molecule has two photoreceptive domains named LOV1 and LOV2 in the N-terminal region. Blue light absorbed by a chromophore in these domains triggers conformational changes in the protein moiety. The C-terminal region of phototropin forms a Ser/Thr kinase that is activated by these conformational changes. The activated phototropin kinase transmits signals downstream leading to tropic responses. The lifetime of the activated state may concern the sensitivity of the tropic responses to light. Thus, spectrophotometric and kinase activity analyses of phototropin are important to understand the light signaling processes related to the photosensitivity. The preparation of polypeptide samples of Arabidopsis phototropin and the methods of spectroscopic measurements and kinase assay of these samples are shown in this chapter.

Photoreaction Dynamics of LOV1 and LOV2 of Phototropin from Chlamydomonas reinhardtii.

Phototropin is a blue light sensor protein found in higher plants and green algae. Photochemical reactions of a variety of differently truncated constructs of a phototropin from Chlamydomonas reinhardtii (Cr) (LOV1, LOV1-hinge, LOV2, LOV2-linker, and hinge-LOV2) are investigated. In the dark state, LOV1 is in dynamic equilibrium between the monomer and dimer, and the main photochemical reaction is dimerization of the monomer and dissociation of the dimer. On the other hand, LOV1-hinge exists as the monomer and the photochemical reaction is the dimerization reaction associated with the unfolding of the helix of the hinge domain. LOV2 in the dark state is monomeric. The conformation changes after the photoexcitation of LOV2 and LOV2-linker are minor, which differs notably from the reaction of LOV2-Jα and LOV2-linker from Arabidopsis thaliana (At). The linker region, including the Jα helix, is rather stable upon photoexcitation. The helix of the hinge domain of hinge-LOV2 is slightly unfolded in the dark state, and the major photoreaction is the dimerization event. The dark recovery rate of LOV2 was found to decrease significantly in the presence of the hinge domain. These photochemical properties of Cr phot are considerably different from those of At phot regarding conformational changes and their kinetics, although Cr phot has been reported to rescue the phot function in At. The differences and the diversity of phots are discussed.

Hydrogen Bonding Environment of the N3-H Group of Flavin Mononucleotide in the Light Oxygen Voltage Domains of Phototropins.

The light oxygen voltage (LOV) domain is a flavin-binding blue-light receptor domain, originally found in a plant photoreceptor phototropin (phot). Recently, LOV domains have been used in optogenetics as the photosensory domain of fusion proteins. Therefore, it is important to understand how LOV domains exhibit light-induced structural changes for the kinase domain regulation, which enables the design of LOV-containing optogenetics tools with higher photoactivation efficiency. In this study, the hydrogen bonding environment of the N3-H group of flavin mononucleotide (FMN) of the LOV2 domain from Adiantum neochrome (neo) 1 was investigated by low-temperature Fourier transform infrared spectroscopy. Using specifically 15N-labeled FMN, [1,3-15N2]FMN, the N3-H stretch was identified at 2831 cm-1 for the unphotolyzed state at 150 K, indicating that the N3-H group forms a fairly strong hydrogen bond. The N3-H stretch showed temperature dependence, with a shift to lower frequencies at ≤200 K and to higher frequencies at ≥250 K from the unphotolyzed to the intermediate states. Similar trends were observed in the LOV2 domains from Arabidopsis phot1 and phot2. By contrast, the N3-H stretch of the Q1029L mutant of neo1-LOV2 and neo1-LOV1 was not temperature dependent in the intermediate state. These results seemed correlated with our previous finding that the LOV2 domains show the structural changes in the ß-sheet region and/or the adjacent Jα helix of LOV2 domain, but that such structural changes do not take place in the Q1029L mutant or neo1-LOV1 domain. The environment around the N3-H group was also investigated.

Light-Induced Conformational Changes of LOV2-Kinase and the Linker Region in Arabidopsis Phototropin2.

Phototropins (phots) are blue light sensors found in a variety of higher plants and algae. The photochemical reactions of this family of proteins have attracted much attention since their discovery. Phots have two light sensor domains called light-oxygen-voltage 1 (LOV1) and LOV2. After the formation of the characteristic adduct of the LOV domain, a conformational change of the C-terminal region of the LOV2 domain occurs, and characterizing this change is important for understanding biological function, that is, kinase activation. Here, the reaction dynamics of the Jα-helix and the extended region adjacent to the Jα-helix (connector) have been investigated. The conformation of the connector part and the Jα-helix were found to alter significantly in a two-state manner. Furthermore, the conformational change of the kinase domain was also successfully detected as a change in translational diffusion, although the CD intensity due to the kinase domain movement was almost silent. These observations indicate that the tertiary structure of the kinase domain changes. The rate of the kinase domain change is almost the same as that of the change for the LOV2-linker, suggesting that the conformational change of the linker is the rate-determining step for kinase activation.

Anomalous pressure effects on the photoreaction of a light-sensor protein from Synechocystis, PixD (Slr1694), and the compressibility change of its intermediates.

SyPixD (Slr1694) is a blue-light receptor that contains a BLUF (blue-light sensor using a flavin chromophore) domain for the function of phototaxis. The key reaction of this protein is a light-induced conformational change and subsequent dissociation reaction from the decamer to the dimer. In this study, anomalous effects of pressure on this reaction were discovered, and changes in the compressibility of its short-lived intermediates were investigated. While the absorption spectra of the dark and light states are not sensitive to pressure, the formation yield of the first intermediate decreases with pressure to about 40% at 150 MPa. Upon blue-light illumination with a sufficiently strong intensity, the transient grating signal, which represents the dissociation of the SyPixD decamer, was observed at 0.1 MPa, and the signal intensity significantly decreased with increasing pressure. This behavior shows that the dissociation of the decamer from the second intermediate state is suppressed by pressure. However, while the decamer undergoes no dissociation upon excitation of one monomer unit at 0.1 MPa, dissociation is gradually induced with increasing pressure. For solving this strange behavior, the compressibility changes of the intermediates were measured as a function of pressure at weak light intensity. Interestingly, the compressibility change was negative at low pressure, but became positive with increasing pressure. Because the compressibility is related to the volume fluctuation, this observation suggests that the driving force for this reaction is fluctuation of the protein. The relationship between the cavities at the interfaces of the monomer units and the reactivity was also discussed.

Time-resolved fluctuation during the photochemical reaction of a photoreceptor protein: phototropin1LOV2-linker.

Although the relationship between structural fluctuations and reactions is important for elucidating reaction mechanisms, experimental data describing such fluctuations of reaction intermediates are sparse. In order to investigate structural fluctuations during a protein reaction, the compressibilities of intermediate species after photoexcitation of a phot1LOV2-linker, which is a typical LOV domain protein with the C-terminal linker including the J-α helix and used recently for optogenetics, were measured in the time-domain by the transient grating and transient lens methods with a high pressure optical cell. The yield of covalent bond formation between the chromophore and a Cys residue (S state formation) relative to that at 0.1 MPa decreased very slightly with increasing pressure. The fraction of the reactive species that yields the T state (linker-unfolded state) decreased almost proportionally with pressure (0.1-200 MPa) to about 65%. Interestingly, the volume change associated with the reaction was much more pressure sensitive. By combining these data, the compressibility changes for the short lived intermediate (S state) and the final product (T state) formation were determined. The compressibility of the S state was found to increase compared with the dark (D) state, and the compressibility decreased during the transition from the S state to the T state. The compressibility change is discussed in terms of cavities inside the protein. By comparing the crystal structures of the phot1LOV2-linker at dark and light states, we concluded that the cavity volumes between the LOV domain and the linker domain increase in the S state, which explains the enhanced compressibility.

The linker between LOV2-Jα and STK plays an essential role in the kinase activation by blue light in Arabidopsis phototropin1, a plant blue light receptor.

Phototropin (phot), a blue light receptor in plants, is composed of several domains: LOV1, LOV2, and a serine/threonine kinase (STK). LOV2 is the main regulator of light activation of STK. However, the detailed mechanism remains unclear. In this report, we focused on the linker region between LOV2 and STK excluding the Jα-helix. Spectroscopy and a kinase assay for the substituents in the linker region of Arabidopsis phot1 LOV2-STK indicated that the linker is involved in the activation of STK. A putative module in the middle of the linker would be critical for intramolecular signaling and/or regulation of STK.

Reconstitution of an Initial Step of Phototropin Signaling in Stomatal Guard Cells.

Phototropins are light-activated receptor kinases that mediate a wide range of blue light responses responsible for the optimization of photosynthesis. Despite the physiological importance of phototropins, it is still unclear how they transduce light signals into physiological responses. Here, we succeeded in reproducing a primary step of phototropin signaling in vitro using a physiological substrate of phototropin, the BLUS1 (BLUE LIGHT SIGNALING1) kinase of guard cells. When PHOT1 and BLUS1 were expressed in Escherichia coli and the resulting recombinant proteins were incubated with ATP, white and blue light induced phosphorylation of BLUS1 but red light and darkness did not. Site-directed mutagenesis of PHOT1 and BLUS1 revealed that the phosphorylation was catalyzed by phot1 kinase. Similar to stomatal blue light responses, the BLUS1 phosphorylation depended on the fluence rate of blue light and was inhibited by protein kinase inhibitors, K-252a and staurosporine. In contrast to the result in vivo, BLUS1 was not dephosphorylated in vitro, suggesting the involvement of a protein phosphatase in the response in vivo. phot1 with a C-terminal kinase domain but devoid of the N-terminal domain, constitutively phosphorylated BLUS1 without blue light, indicating that the N-terminal domain has an autoinhibitory action and prevents substrate phosphorylation. The results provide the first reconstitution of a primary step of phototropin signaling and a clue for understanding the molecular nature of this process.

Essential role of the A'α/Aß gap in the N-terminal upstream of LOV2 for the blue light signaling from LOV2 to kinase in Arabidopsis photototropin1, a plant blue light receptor.

Phototropin (phot) is a blue light (BL) receptor in plants and is involved in phototropism, chloroplast movement, stomata opening, etc. A phot molecule has two photo-receptive domains named LOV (Light-Oxygen-Voltage) 1 and 2 in its N-terminal region and a serine/threonine kinase (STK) in its C-terminal region. STK activity is regulated mainly by LOV2, which has a cyclic photoreaction, including the transient formation of a flavin mononucleotide (FMN)-cysteinyl adduct (S390). One of the key events for the propagation of the BL signal from LOV2 to STK is conformational changes in a Jα-helix residing downstream of the LOV2 C-terminus. In contrast, we focused on the role of the A'α-helix, which is located upstream of the LOV2 N-terminus and interacts with the Jα-helix. Using LOV2-STK polypeptides from Arabidopsis thaliana phot1, we found that truncation of the A'α-helix and amino acid substitutions at Glu474 and Lys475 in the gap between the A'α and the Aß strand of LOV2 (A'α/Aß gap) to Ala impaired the BL-induced activation of the STK, although they did not affect S390 formation. Trypsin digested the LOV2-STK at Lys603 and Lys475 in a light-dependent manner indicating BL-induced structural changes in both the Jα-helix and the gap. The digestion at Lys603 is faster than at Lys475. These BL-induced structural changes were observed with the Glu474Ala and the Lys475Ala substitutes, indicating that the BL signal reached the Jα-helix as well as the A'α/Aß gap but could not activate STK. The amino acid residues, Glu474 and Lys475, in the gap are conserved among the phots of higher plants and may act as a joint to connect the structural changes in the Jα-helix with the activation of STK.

Pressure-sensitive reaction yield of the TePixD blue-light sensor protein.

The effect of pressure on the dissociation reaction of the TePixD decamer was investigated by high-pressure transient grating (TG). The TG signal intensity representing the dissociation reaction of the TePixD decamer significantly decreased by applying a relatively small pressure. On the other hand, the reaction rate increased with increasing pressure. The equilibrium between the pentamer and the decamer was investigated by high-pressure dynamic light scattering. The results indicated that the fraction of the decamer slightly increased in the high-pressure region. From these measurements, it was concluded that the pressure-dependent signal intensity originated from the decrease of the quantum yield of the dissociation reaction of the decamer, indicating that this reaction efficiency is very sensitive to pressure. Using densimetry at high pressures, the compressibility was found to be pressure dependent even in a relatively low pressure range. We attributed the origin of the pressure-sensitive reaction yield to the decrease of compressibility at high pressure. Because the compressibility is related to the volume fluctuation, this observation suggests that the driving force for this reaction is fluctuation of the protein. The relationship between the cavities at the interfaces of the monomer units and the reactivity is also discussed.

The effects of phytochrome-mediated light signals on the developmental acquisition of photoperiod sensitivity in rice.

Plants commonly rely on photoperiodism to control flowering time. Rice development before floral initiation is divided into two successive phases: the basic vegetative growth phase (BVP, photoperiod-insensitive phase) and the photoperiod-sensitive phase (PSP). The mechanism responsible for the transition of rice plants into their photoperiod-sensitive state remains elusive. Here, we show that se13, a mutation detected in the extremely early flowering mutant X61 is a nonsense mutant gene of OsHY2, which encodes phytochromobilin (PΦB) synthase, as evidenced by spectrometric and photomorphogenic analyses. We demonstrated that some flowering time and circadian clock genes harbor different expression profiles in BVP as opposed to PSP, and that this phenomenon is chiefly caused by different phytochrome-mediated light signal requirements: in BVP, phytochrome-mediated light signals directly suppress Ehd2, while in PSP, phytochrome-mediated light signals activate Hd1 and Ghd7 expression through the circadian clock genes' expression. These findings indicate that light receptivity through the phytochromes is different between two distinct developmental phases corresponding to the BVP and PSP in the rice flowering process. Our results suggest that these differences might be involved in the acquisition of photoperiod sensitivity in rice.

Transient conformational fluctuation of TePixD during a reaction.

Knowledge of the dynamical behavior of proteins, and in particular their conformational fluctuations, is essential to understanding the mechanisms underlying their reactions. Here, transient enhancement of the isothermal partial molar compressibility, which is directly related to the conformational fluctuation, during a chemical reaction of a blue light sensor protein from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 (TePixD, Tll0078) was investigated in a time-resolved manner. The UV-Vis absorption spectrum of TePixD did not change with the application of high pressure. Conversely, the transient grating signal intensities representing the volume change depended significantly on the pressure. This result implies that the compressibility changes during the reaction. From the pressure dependence of the amplitude, the compressibility change of two short-lived intermediate (I1 and I2) states were determined to be +(5.6 ± 0.6) × 10(-2) cm(3) ⋅ mol(-1) ⋅ MPa(-1) for I1 and +(6.6 ± 0.7) × 10(-2) cm(3) ⋅ mol(-1) ⋅ MPa(-1) for I2. This result showed that the structural fluctuation of intermediates was enhanced during the reaction. To clarify the relationship between the fluctuation and the reaction, the compressibility of multiply excited TePixD was investigated. The isothermal compressibility of I1 and I2 intermediates of TePixD showed a monotonic decrease with increasing excitation laser power, and this tendency correlated with the reactivity of the protein. This result indicates that the TePixD decamer cannot react when its structural fluctuation is small. We concluded that the enhanced compressibility is an important factor for triggering the reaction of TePixD. To our knowledge, this is the first report showing enhanced fluctuations of intermediate species during a protein reaction, supporting the importance of fluctuations.

A dominant mutation in the light-oxygen and voltage2 domain vicinity impairs phototropin1 signaling in tomato.

In higher plants, blue light (BL) phototropism is primarily controlled by the phototropins, which are also involved in stomatal movement and chloroplast relocation. These photoresponses are mediated by two phototropins, phot1 and phot2. Phot1 mediates responses with higher sensitivity than phot2, and phot2 specifically mediates chloroplast avoidance and dark positioning responses. Here, we report the isolation and characterization of a Nonphototropic seedling1 (Nps1) mutant of tomato (Solanum lycopersicum). The mutant is impaired in low-fluence BL responses, including chloroplast accumulation and stomatal opening. Genetic analyses show that the mutant locus is dominant negative in nature. In dark-grown seedlings of the Nps1 mutant, phot1 protein accumulates at a highly reduced level relative to the wild type and lacks BL-induced autophosphorylation. The mutant harbors a single glycine-1484-to-alanine transition in the Hinge1 region of a phot1 homolog, resulting in an arginine-to-histidine substitution (R495H) in a highly conserved A'α helix proximal to the light-oxygen and voltage2 domain of the translated gene product. Significantly, the R495H substitution occurring in the Hinge1 region of PHOT1 abolishes its regulatory activity in Nps1 seedlings, thereby highlighting the functional significance of the A'α helix region in phototropic signaling of tomato.

Light-induced conformational changes of LOV1 (light oxygen voltage-sensing domain 1) and LOV2 relative to the kinase domain and regulation of kinase activity in Chlamydomonas phototropin.

Phototropin (phot), a blue light (BL) receptor in plants, has two photoreceptive domains named LOV1 and LOV2 as well as a Ser/Thr kinase domain (KD) and acts as a BL-regulated protein kinase. A LOV domain harbors a flavin mononucleotide that undergoes a cyclic photoreaction upon BL excitation via a signaling state in which the inhibition of the kinase activity by LOV2 is negated. To understand the molecular mechanism underlying the BL-dependent activation of the kinase, the photochemistry, kinase activity, and molecular structure were studied with the phot of Chlamydomonas reinhardtii. Full-length and LOV2-KD samples of C. reinhardtii phot showed cyclic photoreaction characteristics with the activation of LOV- and BL-dependent kinase. Truncation of LOV1 decreased the photosensitivity of the kinase activation, which was well explained by the fact that the signaling state lasted for a shorter period of time compared with that of the phot. Small angle x-ray scattering revealed monomeric forms of the proteins in solution and detected BL-dependent conformational changes, suggesting an extension of the global molecular shapes of both samples. Constructed molecular model of full-length phot based on the small angle x-ray scattering data proved the arrangement of LOV1, LOV2, and KD for the first time that showed a tandem arrangement both in the dark and under BL irradiation. The models suggest that LOV1 alters its position relative to LOV2-KD under BL irradiation. This finding demonstrates that LOV1 may interact with LOV2 and modify the photosensitivity of the kinase activation through alteration of the duration of the signaling state in LOV2.

Dynamics of the amino-terminal and carboxyl-terminal helices of Arabidopsis phototropin 1 LOV2 studied by the transient grating.

Recently, conformational changes of the amino-terminal helix (A'α helix), in addition to the reported conformational changes of the carboxyl-terminal helix (Jα helix), have been proposed to be important for the regulatory function of the light-oxygen-voltage 2 domain (LOV2) of phototropin 1 from Arabidopsis. However, the reaction dynamics of the A'α helix have not been examined. Here, the unfolding reactions of the A'α and Jα helices of the LOV2 domain of phototropin 1 from Arabidopsis thaliana were investigated by the time-resolved transient grating (TG) method. A mutant (T469I mutant) that renders the A'α helix unfolded in the dark state showed unfolding of the Jα helix with a time constant of 1 ms, which is very similar to the time constant reported for the wild-type LOV2-linker sample. Furthermore, a mutant (I608E mutant) that renders the Jα helix unfolded in the dark state exhibited an unfolding process of the A'α helix with a time constant of 12 ms. On the basis of these experimental results, it is suggested that the unfolding reactions of these helices occurs independently.

Photochemistry of Arabidopsis phototropin 1 LOV1: transient tetramerization.

The photochemical reaction of the LOV1 (light-oxygen-voltage 1) domain of phototropin 1 from Arabidopsis thaliana was investigated by the time-resolved transient grating method. As with other LOV domains, an absorption spectral change associated with an adduct formation between its chromophore (flavin mononucleotide) and a cysteine residue was observed with a time constant of 1.1 µs. After this reaction, a significant diffusion coefficient (D) change (D of the reactant = 8.2 × 10(-11) m(2) s(-1), and D of the photoproduct = 6.4 × 10(-11) m(2) s(-1)) was observed with a time constant of 14 ms at a protein concentration of 270 µM. From the D value of the ground state and the peak position in size exclusion chromatography, we have confirmed that the phot1LOV1 domain exists as a dimer in the dark. The D-value and the concentration dependence of the rate indicated that the phot1LOV1 domain associates to form a tetramer (dimerization of the dimer) upon photoexcitation. We also found that the chromophore is released from the binding pocket of the LOV domain when it absorbs two photons within a pulse duration, which occurs in addition to the normal photocycle reaction. On the basis of these results, we discuss the molecular mechanism of the light dependent role of the phot1LOV1 domain.

Anomalous diffusion of TePixD and identification of the photoreaction product.

TePixD is a blue-light sensor protein from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 (TePixD Tll0078). Although the photochemistry has been examined, so far the photoproduct remains unknown. We have measured the diffusion coefficient (D) of TePixD in the dark by dynamic light scattering and have discovered a very peculiar diffusion property; the decamer oligomer has a larger D than that of the pentamer. Furthermore, D of the pentamer was found to be very close to that of the TePixD decamer photoreaction product. In order to investigate this reaction further, elution profiles of size-exclusion chromatography were measured under dark and illuminated conditions at low (40 µM) and high (1.1 mM) TePixD concentrations. On the basis of these results, we have concluded that the main photoreaction of the TePixD decamer is the dissociation into the pentamer. The secondary structure change associated with this reaction was found to be minor according to circular dichroism analysis.