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Chloroplasts accumulate in regions of plant cells exposed to irradiation to maximize light reception for efficient photosynthesis. This response is mediated by the blue-light receptor phototropin. Upon the perception of blue light, phototropin is photoactivated, an unknown signal is transmitted from the photoactivated phototropin to distant chloroplasts, and the chloroplasts begin their directional movement. How activated phototropin initiates this signal transmission is unknown. Here, using the liverwort Marchantia polymorpha, we analysed whether increased photoactive phototropin levels mediate signal transmission and chloroplast behaviour during the accumulation response. The signal transmission rate was higher in transgenic cells overexpressing phototropin than in wild-type cells. However, the chloroplast directional movement was similar between wild-type and transgenic cells. Consistent with the observation, increasing the amount of photoactivated phototropin through higher blue-light intensity also accelerated signal transmission but did not affect chloroplast behaviour in wild-type cells. Photoactivation of phototropin under weak blue-light led to the greater protein level of phosphorylated phototropin in cells overexpressing phototropin than in wild-type cells, whereas the autophosphorylation level within each phototropin molecule was similar. These results indicate that the abundance of photoactivated phototropin modulates the signal transmission rate to distant chloroplasts but does not affect chloroplast behaviour during the accumulation response., (© 2024 John Wiley & Sons Ltd.)

Key Message: Brachypodium distachyon is a good model for studying chloropla st movements in the crop plants, wheat, rye and barley. The movements are activated only by blue light, similar to Arabidopsis. Chloroplast translocations are ubiquitous in photosynthetic organisms. On the one hand, they serve to optimize energy capture under limiting light, on the other hand, they minimize potential photodamage to the photosynthetic apparatus in excess light. In higher plants chloroplast movements are mediated by phototropins (phots), blue light receptors that also control other light acclimation responses. So far, Arabidopsis thaliana has been the main model for studying the mechanism of blue light signaling to chloroplast translocations in terrestrial plants. Here, we propose Brachypodium distachyon as a model in research into chloroplast movements in C3 cereals. Brachypodium chloroplasts respond to light in a similar way to those in Arabidopsis. The amino acid sequence of Brachypodium PHOT1 is 79.3% identical, and that of PHOT2 is 73.6% identical to the sequence of the corresponding phototropin in Arabidopsis. Both phototropin1 and 2 are expressed in Brachypodium, as shown using quantitative real-time PCR. Intriguingly, the light-expression pattern of BradiPHOT1 and BradiPHOT2 is the opposite of that for Arabidopsis phototropins, suggesting potential unique light signaling in C3 grasses. To investigate if Brachypodium is a good model for studying grass chloroplast movements we analyzed these movements in the leaves of three C3 crop grasses, namely wheat, rye and barley. Similarly to Brachypodium, chloroplasts only respond to blue light in all these species.

LOV (light-, oxygen- or voltage-sensitive) domains act as photosensory units of many prokaryotic and eukaryotic proteins. Upon blue light excitation they undergo a photocycle via the excited triplet state of their flavin chromophore yielding the flavin-cysteinyl adduct. Adduct formation is highly conserved among all LOV domains and constitutes the primary step of LOV domain signaling. But recently, it has been shown that signal propagation can also be triggered by flavin photoreduction to the neutral semiquinone offering new prospects for protein engineering. This, however, requires mutation of the photo-active Cys. Here, we report on LOV1 mutants of C. reinhardtii phototropin in which adduct formation is suppressed although the photo-active Cys is present. Introduction of a Tyr into the LOV core induces a proton coupled electron transfer towards the flavin chromophore. Flavin radical species are formed via either the excited flavin singlet or triplet state depending on the geometry of donor and acceptor. This photoreductive pathway resembles the photoreaction observed in other blue light photoreceptors, e.g. blue-light sensors using flavin adenine dinucleotide (BLUF) domains or cryptochromes. The ability to tune the photoreactivity of the flavin chromophore inside the LOV core has implications for the mechanism of adduct formation in the wild type and may be of use for protein engineering.

The rational engineering of photosensor proteins underpins the field of optogenetics, in which light is used for spatiotemporal control of cell signaling. Optogenetic elements function by converting electronic excitation of an embedded chromophore into structural changes on the microseconds to seconds time scale, which then modulate the activity of output domains responsible for biological signaling. Using time-resolved vibrational spectroscopy coupled with isotope labeling, we have mapped the structural evolution of the LOV2 domain of the flavin binding phototropin Avena sativa (AsLOV2) over 10 decades of time, reporting structural dynamics between 100 fs and 1 ms after optical excitation. The transient vibrational spectra contain contributions from both the flavin chromophore and the surrounding protein matrix. These contributions are resolved and assigned through the study of four different isotopically labeled samples. High signal-to-noise data permit the detailed analysis of kinetics associated with the light activated structural evolution. A pathway for the photocycle consistent with the data is proposed. The earliest events occur in the flavin binding pocket, where a subpicosecond perturbation of the protein matrix occurs. In this perturbed environment, the previously characterized reaction between triplet state isoalloxazine and an adjacent cysteine leads to formation of the adduct state; this step is shown to exhibit dispersive kinetics. This reaction promotes coupling of the optical excitation to successive time-dependent structural changes, initially in the β-sheet and then α-helix regions of the AsLOV2 domain, which ultimately gives rise to Jα-helix unfolding, yielding the signaling state. This model is tested through point mutagenesis, elucidating in particular the key mediating role played by Q513.

In plants and algae, light serves both as the energy source for photosynthesis and a biological signal that triggers cellular responses via specific sensory photoreceptors. Red light is perceived by bilin-containing phytochromes and blue light by the flavin-containing cryptochromes and/or phototropins (PHOTs), the latter containing two photosensory light, oxygen, or voltage (LOV) domains. Photoperception spans several orders of light intensity, ranging from far below the threshold for photosynthesis to values beyond the capacity of photosynthetic CO 2 assimilation. Excess light may cause oxidative damage and cell death, processes prevented by enhanced thermal dissipation via high-energy quenching (qE), a key photoprotective response. Here we show the existence of a molecular link between photoreception, photosynthesis, and photoprotection in the green alga Chlamydomonas reinhardtii. We show that PHOT controls qE by inducing the expression of the qE effector protein LHCSR3 (light-harvesting complex stress-related protein 3) in high light intensities. This control requires blue-light perception by LOV domains on PHOT, LHCSR3 induction through PHOT kinase, and light dissipation in photosystem II via LHCSR3. Mutants deficient in the PHOT gene display severely reduced fitness under excessive light conditions, indicating that the sensing, utilization, and dissipation of light is a concerted process that plays a vital role in microalgal acclimation to environments of variable light intensities.

Background: Regulated proteolysis by the proteasome is one of the fundamental mechanisms used in eukaryotic cells to control cellular behavior. Efficient tools to regulate protein stability offer synthetic influence on molecular level on a selected biological process. Optogenetic control of protein stability has been achieved with the photo-sensitive degron (psd) module. This engineered tool consists of the photoreceptor domain light oxygen voltage 2 (LOV2) from Arabidopsis thaliana phototropin1 fused to a sequence that induces direct proteasomal degradation, which was derived from the carboxy-terminal degron of murine ornithine decarboxylase. The abundance of target proteins tagged with the psd module can be regulated by blue light if the degradation tag is exposed to the cytoplasm or the nucleus., Results: We used the model organism Saccharomyces cerevisiae to generate psd module variants with increased and decreased stabilities in darkness or when exposed to blue light using site-specific and random mutagenesis. The variants were characterized as fusions to fluorescent reporter proteins and showed half-lives between 6 and 75 minutes in cells exposed to blue light and 14 to 187 minutes in darkness. In blue light, ten variants showed accelerated degradation and four variants increased stability compared to the original psd module. Measuring the dark/light ratio of selected constructs in yeast cells showed that two variants were obtained with ratios twice as high as in the wild type psd module. In silico modeling of photoreceptor variant characteristics suggested that for most cases alterations in behavior were induced by changes in the light-response of the LOV2 domain., Conclusions: In total, the mutational analysis resulted in psd module variants, which provide tuning of protein stability over a broad range by blue light. Two variants showed characteristics that are profoundly improved compared to the original construct. The modular usage of the LOV2 domain in optogenetic tools allows the usage of the mutants in the context of other applications in synthetic and systems biology as well.

The function of many eukaryotic proteins is regulated by highly dynamic changes in their nucleocytoplasmic distribution. The ability to precisely and reversibly control nuclear translocation would, therefore, allow dissecting and engineering cellular networks. Here we develop a genetically encoded, light-inducible nuclear localization signal (LINuS) based on the LOV2 domain of Avena sativa phototropin 1. LINuS is a small, versatile tag, customizable for different proteins and cell types. LINuS-mediated nuclear import is fast and reversible, and can be tuned at different levels, for instance, by introducing mutations that alter AsLOV2 domain photo-caging properties or by selecting nuclear localization signals (NLSs) of various strengths. We demonstrate the utility of LINuS in mammalian cells by controlling gene expression and entry into mitosis with blue light.

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.

Genetic studies have shown the effects of various photoreceptors on early photomorphogenic processes, defining the precise time course of red (RL), far-red (FrL) and blue light (BL) action. In this study, the effect of green wavebands in conjunction with these responses is examined. Longer-term (end point; 24-96 h) analysis of hypocotyl elongation in enriched green environments shows an increase in growth compared to seedlings under blue, red or both together. The effect was only observed at lower fluence rates (<10 μmol/m² s). Genetic analyses demonstrate that cryptochromes are required for this GL effect, consistent with earlier findings, and that the phy receptors have no influence. However, analysis of early (minutes to hours) stem growth kinetics indicates that GL cannot reverse the cryptochrome-mediated BL effect during early stem growth inhibition, and instead acts additively with BL to drive cryptochrome-mediated inhibition. Green light (GL) treatments antagonize RL and FrL-mediated hypocotyl inhibition. The GL opposition of RL responses persists in phyA, phyB, cry1cry2 and phot2 mutants. The response requires phot1 and NPH3, suggesting that this is not a GL response, but instead a response to extremely low-fluence rate BL. Tests with dim BL (<0.1 μmol/m² s) confirm a previously uncharacterized phot1-dependent promotion of stem growth, opposing the effects of RL. These findings demonstrate how enriched green environments may adjust RL and BL photomorphogenic responses through both the crys and phot1 receptors, and define a new role for phot1 in stem growth promotion.

The eyespot of Chlamydomonas reinhardtii is a light-sensitive organelle important for phototactic orientation of the alga. Here, we found that eyespot size is strain specific and downregulated in light. In a strain in which the blue light photoreceptor phototropin was deleted by homologous recombination, the light regulation of the eyespot size was affected. We restored this dysfunction in different phototropin complementation experiments. Complementation with the phototropin kinase fragment reduced the eyespot size, independent of light. Interestingly, overexpression of the N-terminal light, oxygen or voltage sensing domains (LOV1+LOV2) alone also affected eyespot size and phototaxis, suggesting that aside from activation of the kinase domain, they fulfill an independent signaling function in the cell. Moreover, phototropin is involved in adjusting the level of channelrhodopsin-1, the dominant primary receptor for phototaxis within the eyespot. Both the level of channelrhodopsin-1 at the onset of illumination and its steady state level during the light period are downregulated by phototropin, whereas the level of channelrhodopsin-2 is not significantly altered. Furthermore, a light intensity-dependent formation of a C-terminal truncated phototropin form was observed. We propose that phototropin is a light regulator of phototaxis that desensitizes the eyespot when blue light intensities increase.

Naturally photoswitchable proteins offer a means of directly manipulating the formation of protein complexes that drive a diversity of cellular processes. We developed tunable light-inducible dimerization tags (TULIPs) based on a synthetic interaction between the LOV2 domain of Avena sativa phototropin 1 (AsLOV2) and an engineered PDZ domain (ePDZ). TULIPs can recruit proteins to diverse structures in living yeast and mammalian cells, either globally or with precise spatial control using a steerable laser. The equilibrium binding and kinetic parameters of the interaction are tunable by mutation, making TULIPs readily adaptable to signaling pathways with varying sensitivities and response times. We demonstrate the utility of TULIPs by conferring light sensitivity to functionally distinct components of the yeast mating pathway and by directing the site of cell polarization.

Signaling networks in living systems are coordinated through subcellular compartmentalization and precise timing of activation. These spatiotemporal aspects ensure the fidelity of signaling while contributing to the diversity and specificity of downstream events. This is studied through development of molecular tools that generate localized and precisely timed protein activity in living systems. To study the molecular events responsible for cytoskeletal changes in real time, we generated versions of Rho family GTPases whose interactions with downstream effectors is controlled by light. GTPases were grafted to the phototropin LOV (light, oxygen, or voltage) domain (Huala, E., Oeller, P. W., Liscum, E., Han, I., Larsen, E., and Briggs, W. R. (1997). Arabidopsis NPH1: A protein kinase with a putative redox-sensing domain. Science278, 2120-2123.) via an alpha helix on the LOV C-terminus (Wu, Y. I., Frey, D., Lungu, O. I., Jaehrig, A., Schlichting, I., Kuhlman, B., and Hahn, K. M. (2009). A genetically encoded photoactivatable Rac controls the motility of living cells. Nature461, 104-108.). The LOV domain sterically blocked the GTPase active site until it was irradiated. Exposure to 400-500nm light caused unwinding of the helix linking the LOV domain to the GTPase, relieving steric inhibition. The change was reversible and repeatable, and the protein could be returned to its inactive state simply by turning off the light. The LOV domain incorporates a flavin as the active chromophore. This naturally occurring molecule is incorporated simply upon expression of the LOV fusion in cells or animals, permitting ready control of GTPase function in different systems. In cultured single cells, light-activated Rac leads to membrane ruffling, protrusion, and migration. In collectively migrating border cells in the Drosophila ovary, focal activation of photoactivatable Rac (PA-Rac) in a single cell is sufficient to redirect the entire group. PA-Rac in a single cell also rescues the phenotype caused by loss of endogenous guidance receptor signaling in the whole group. These findings demonstrate that cells within the border cell cluster communicate and are guided collectively. Here, we describe optimization and application of PA-Rac using detailed examples that we hope will help others apply the approach to different proteins and in a variety of different cells, tissues, and organisms., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The blue-light photoreceptor phototropin plays a crucial role in optimizing photosynthesis in plants. In the two light-, oxygen-, or voltage-sensitive (LOV) domains of phototropin, the light stimulus is absorbed by the flavin chromophores. The signal is assumed to be transferred via dissociation and unfolding of a conserved J alpha helix element to the serine/threonine kinase domain. We investigated full-length phototropin from the green alga Chlamydomonas reinhardtii by Fourier transform infrared spectroscopy to shed light on the signal transfer within the protein and on the structural response of the kinase. Light-induced structural changes were assigned by comparing signals of the full-length protein with those of the truncated LOV1-LOV2-J alpha and LOV1-LOV2 and with those of deletion mutants. A loss of helicity originating from the J alpha linker helix was observed in LOV1-LOV2-J alpha in agreement with previous studies of LOV2-J alpha. Full-length phototropin showed reversible global conformational changes via several turn elements. These changes were suppressed in a deletion mutant lacking the J alpha linker and are attributed to the kinase domain. The loss of turn structure is interpreted as a light-induced opening of the kinase tertiary structure upon release of the LOV2 domain. Concomitant protonation changes of Asp or Glu residues in the kinase domain were not observed. A light-induced loss in helicity was observed only in the presence of a phototropin-characteristic 54-amino acid extension of the kinase activation loop, which is predicted to be located apart from the catalytic cleft. This response of the extension might play a significant role in the phototropin signaling process.

Phototropins (phot1 and phot2) are plasma membrane-associated receptor kinases that respond specifically to blue and UV wavelengths. In addition to a C-terminal Ser/Thr kinase domain, phototropins contain two N-terminal chromophore binding LOV domains that function as photoswitches to regulate a wide range of enzymatic activities in prokaryotes and eukaryotes. Through domain swapping, we show that the photochemical properties of Arabidopsis thaliana phot1 rely on interactions between LOV1 and LOV2, which are facilitated by their intervening linker sequence. Functional analysis of domain-swap proteins supports a mechanism whereby LOV2 acts as a dark-state repressor of phot1 activity both in vitro and in vivo. Moreover, we find a photoactive role for LOV1 in arresting chloroplast accumulation at high light intensities. Unlike LOV2, LOV1 cannot operate as a dark-state repressor, resulting in constitutive receptor autophosphorylation and accelerated internalization from the plasma membrane. Coexpression of active and inactive forms of phot1 demonstrates that autophosphorylation can occur intermolecularly, independent of LOV1, via light-dependent receptor dimerization in vivo. Indeed, transphosphorylation is sufficient to promote phot1 internalization through a clathrin-dependent endocytic pathway triggered primarily by phosphorylation of Ser-851 within the kinase activation loop. The mechanistic implications of these findings in regard to light-driven receptor activation and trafficking are discussed.

Phototropin 1 (phot1) is a photoreceptor for phototropism, chloroplast movement, stomatal opening, leaf expansion, and solar tracking in response to blue light. Following earlier work with PHOT1::GFP (Sakamoto and Briggs, 2002), we investigated the pattern of cellular and subcellular localization of phot1 in 3- 4-d-old etiolated seedlings of Arabidopsis thalinana. As expressed from native upstream sequences, the PHOT1::GFP fusion protein is expressed strongly in the abaxial tissues of the cotyledons and in the elongating regions of the hypocotyl. It is moderately expressed in the shoot/root transition zone and in cells near the root apex. A fluorescence signal is undetectable in the root epidermis, root cap, and root apical meristem itself. The plasma membranes of mesophyll cells near the cotyledon margin appear labeled uniformly but cross-walls created by recent cell divisions are more strongly labeled. The pattern of labeling of individual cell types varies with cell type and developmental stage. Blue-light treatment causes PHOT1::GFP, initially relatively evenly distributed at the plasma membrane, to become reorganized into a distinct mosaic with strongly labeled punctate areas and other areas completely devoid of fluorescence--a phenomenon best observed in cortical cells in the hypocotyl elongation region. Concomitant with or following this reorganization, PHOT1::GFP moves into the cytoplasm in all cell types investigated except for guard cells. It disappears from the cytoplasm by an unidentified mechanism after several hours in darkness. Neither its appearance in the cytoplasm nor its eventual disappearance in darkness is prevented by the translation inhibitor cycloheximide, although the latter process is retarded. We hypothesize that blue-light-induced phot1 re-localization modulates blue-light-activated signal transduction.