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Optogenetic tools have been proven to be useful in regulating cellular processes via an external signal. Light can be applied with high spatial and temporal precision as well as easily modulated in quantity and quality. Natural photoreceptors of the light oxygen voltage (LOV) domain family have been characterized in depth, especially the LOV2 domain of Avena sativa (As) phototropin 1 and its derivatives. Information on the behavior of LOV2 variants with changes in the photocycle or the light response has been recorded. Here, we applied well-described photocycle mutations on the AsLOV2 domain of a photosensitive transcription factor (psTF) as well as its variant that is part of the photosensitive degron (psd) psd3 in Saccharomyces cerevisiae. In vivo and in vitro measurements revealed that each photoreceptor component of the light-sensitive transcription factor and the psd3 module can be modulated in its light sensitivity by mutations that are known to prolong or shorten the dark-reversion time of AsLOV2. Yet, only two of the mutations showed differences in the in vivo behavior in the context of the psd3 module. For the AsLOV2 domain in the context of the psTF, we observed different characteristics for all four variants. Molecular dynamics simulations showed distinct influences of the shortened Jα helix and the V416L mutation in the context of the psd3 photoreceptor. In conclusion, we demonstrated the tunability of two optogenetic tools with a set of mutations that affect the photocycle of the inherent photoreceptors. As these optogenetic tools are concurrent in their action, pleiotropic effects on target protein abundance are achievable with the simultaneous action of the diverse photoreceptor variants.

Polarity underlies all directional growth responses in plants including growth towards the light (phototropism). The plasma-membrane associated protein, NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3) is a key determinant of phototropic growth which is regulated by phototropin (phot) AGC kinases. Here we demonstrate that NPH3 is directly phosphorylated by phot1 within a conserved C-terminal consensus sequence (RxS) that is necessary to promote phototropism and petiole positioning in Arabidopsis. RxS phosphorylation also triggers 14-3-3 binding combined with changes in NPH3 phosphorylation and localisation status. Mutants of NPH3 that are unable to bind or constitutively bind 14-3-3 s show compromised functionality consistent with a model where phototropic curvature is established by signalling outputs arising from a gradient of NPH3 RxS phosphorylation across the stem. Our findings therefore establish that NPH3/RPT2-Like (NRL) proteins are phosphorylation targets for plant AGC kinases. Moreover, RxS phosphorylation is conserved in other members of the NRL family, suggesting a common mechanism of regulating plant growth to the prevailing light environment., The plasma membrane-localized NPH3 protein is required for phototropic growth in Arabidopsis. Here the authors show that phototropin 1 phosphorylates NPH3 at a conserved C-terminal sequence motif triggering binding of 14-3-3 proteins to NPH3 that is necessary for the phototropic response.

Hypocotyl phototropism is redundantly mediated by phot1 and phot2, two blue light receptor phototropins, under the intensity of blue light>1 μmol m−2 s−1. As light intensity increases, phot1 inhibits the phot2-mediated response. To date, only Arabidopsis Root Phototropism2 (RPT2) has been shown to participate in phot1-mediated inhibition of phototropism. To dissect the signaling network that underlies phot1-mediated inhibition, we carried out a yeast two-hybrid (Y2H) screening assay for RPT2 interacting proteins and identified J-domain protein required for chloroplast accumulation response 1 (JAC1). The interaction between JAC1 and RPT2 was verified by bimolecular fluorescence complementation and Co-IP assays. JAC1 is expressed mainly in cotyledons and hypocotyls. Like RPT2, JAC1 can be induced by blue light, suggesting that it may function similarly to RPT2 in the inhibition of phototropism. Genetic analysis showed that jac1 mutation significantly enhanced the hypocotyl bending of phot1 mutants towards intermediate-intensity blue light, and this effect was inhibited by the constitutive expression of JAC1 in the phot1 jac1 mutant. The phot1 rpt2 double mutant also exhibited enhanced phototropism compared with the phot1 mutant. Taken together, our data clearly demonstrate that JAC1 cooperates with RPT2 to negatively regulate hypocotyl phototropism in plants and may act either downstream of or in parallel with phot1.

Plants growing at high densities interact via a multitude of pathways. Here, we provide an overview of mechanisms and functional consequences of plant architectural responses initiated by light cues that occur in dense vegetation. We will review the current state of knowledge about shade avoidance, as well as its possible applications. On an individual level, plants perceive neighbour‐associated changes in light quality and quantity mainly with phytochromes for red and far‐red light and cryptochromes and phototropins for blue light. Downstream of these photoreceptors, elaborate signalling and integration takes place with the PHYTOCHROME INTERACTING FACTORS, several hormones and other regulators. This signalling leads to the shade avoidance responses, consisting of hyponasty, stem and petiole elongation, apical dominance and life cycle adjustments. Architectural changes of the individual plant have consequences for the plant community, affecting canopy structure, species composition and population fitness. In this context, we highlight the ecological, evolutionary and agricultural importance of shade avoidance., Causes and consequences of plant–plant signalling through light cues: from genes to canopies.

Light-activated protein domains provide a convenient, modular, and genetically encodable sensor for optogenetics and optobiology. Although these domains have now been deployed in numerous systems, the precise mechanism of photoactivation and the accompanying structural dynamics that modulate output domain activity remain to be fully elucidated. In the C-terminal light, oxygen, voltage (LOV) domain of plant phototropins (LOV2), blue light activation leads to formation of an adduct between a conserved Cys residue and the embedded FMN chromophore, rotation of a conserved Gln (Q513), and unfolding of a helix (Jα-helix) which is coupled to the output partner. In the present work, we focus on the allosteric pathways leading to Jα helix unfolding in Avena sativa LOV2 (AsLOV2) using an interdisciplinary approach involving molecular dynamics simulations extending to 7 μs, time-resolved infrared spectroscopy, solution NMR spectroscopy, and in-cell optogenetic experiments. In the dark state, the side chain of N414 is hydrogen bonded to the backbone N-H of Q513. The simulations predict a lever-like motion of Q513 after Cys adduct formation resulting in loss of the interaction between the side chain of N414 and the backbone C=O of Q513, and formation of a transient hydrogen bond between the Q513 and N414 side chains. The central role of N414 in signal transduction was evaluated by site-directed mutagenesis supporting a direct link between Jα helix unfolding dynamics and the cellular function of the Zdk2-AsLOV2 optogenetic construct. Through this multifaceted approach, we show that Q513 and N414 are critical mediators of protein structural dynamics, linking the ultrafast (sub-ps) excitation of the FMN chromophore to the microsecond conformational changes that result in photoreceptor activation and biological function.

Light is a pivotal environmental element capable of influencing multiple physiological processes across the entire plant life cycle. Over the course of their evolution, plants have developed several families of photoreceptors such as phytochromes, phototropins, ultraviolet (UV) resistance locus 8 and cryptochromes (crys), in order to sense light stimuli and respond to their changes. Numerous genetic studies have demonstrated that functional alterations to these photoreceptors cause a change in important agronomical traits. In particular, crys, which absorb UVA/blue light, can influence seed germination, flowering induction, plant architecture, fruit metabolic content and resistance to biotic and abiotic stresses. In the years to come, the rising temperatures and alterations to precipitation patterns generated by climate change will present a dramatic challenge for our agricultural system, with its few varieties characterized by a narrow genetic pool derived from artificial selection. Here, we review the main roles of crys in determining important agronomic traits in crops, we discuss the opportunities of using these photoreceptors as genetic targets for tuning plant physiological responses to environmental change, and the molecular strategies used so far to manipulate this family of photoreceptors.

One conserved feature among angiosperms is the development of flat thin leaves. This developmental pattern optimizes light capture and gas exchange. The blue light (BL) receptors phototropins are required for leaf flattening, with the null phot1phot2 mutant showing curled leaves in Arabidopsis (Arabidopsis thaliana). However, key aspects of their function in leaf development remain unknown. Here, we performed a detailed spatiotemporal characterization of phototropin function in Arabidopsis leaves. We found that phototropins perceive light direction in the blade, and, similar to their role in hypocotyls, they control the spatial pattern of auxin signaling, possibly modulating auxin transport, to ultimately regulate cell expansion. Phototropin signaling components in the leaf partially differ from hypocotyls. Moreover, the light response on the upper and lower sides of the leaf blade suggests a partially distinct requirement of phototropin signaling components on each side. In particular, NON PHOTOTROPIC HYPOCOTYL 3 showed an adaxial-specific function. In addition, we show a prominent role of PHYTOCHROME KINASE SUBSTRATE 3 in leaf flattening. Among auxin transporters, PIN-FORMED 3,4,7 and AUXIN RESISTANT 1 (AUX1)/LIKE AUXIN RESISTANT 1 (LAX1) are required for the response while ABCB19 has a regulatory role. Overall, our results show that directional BL perception by phototropins is a key aspect of leaf development, integrating endogenous and exogenous signals., Phototropins perceive light direction in the leaf and control the auxin signaling pattern to regulate blade flattening.

Premise While essential for photosynthesis, excess light can damage plants. We investigated how growth light conditions affect two photoprotective strategies, chloroplast movement and nonphotochemical quenching (NPQ), as well as electron transport rates (ETR), and the relative importance of these processes in the short-term stress tolerance of Arabidopsis thaliana. Methods We grew wild-type (WT) and mutant plants with impaired chloroplast movement (phot1, phot2, phot1 phot2, chup1) or NPQ (npq1) at low (160 μmol photons m-2 s-1 ) or intermediate light (400 μmol photons m-2 s-1 ) before quantifying transmission changes due to chloroplast movement, NPQ, ETR, and the ability to recover from a short-term high-light treatment. Results Plants with impaired chloroplast avoidance movement (phot2, phot1 phot2, chup1) did not recover as well from a short-term high light treatment as the WT or npq1 and phot1 mutants. Plants grown at intermediate light recovered more completely from the same stress treatment regardless of their genotype and despite reduced degrees of transmission changes due to chloroplast movement. This result was due in part to all genotypes having up to a 2-fold increase in ETRmax and a slight increase in NPQmax . Conclusions Growth light conditions affect which mechanisms are important in dealing with short-term high-light stress. The chloroplast avoidance response is important for low-light-grown plants, while increases in ETRmax and NPQmax allow plants grown at intermediate light intensities to avoid being damaged.

Optogenetic techniques use light-responsive proteins to study dynamic processes in living cells and organisms. These techniques typically rely on repurposed naturally occurring light-sensitive proteins to control sub-cellular localization and activity. We previously engineered two optogenetic systems, the Light Activated Nuclear Shuttle (LANS) and the Light-Inducible Nuclear eXporter (LINX), by embedding nuclear import or export sequence motifs into the C-terminal helix of the light-responsive LOV2 domain of Avena sativa phototropin 1, thus enabling light-dependent trafficking of a target protein into and out of the nucleus. While LANS and LINX are effective tools, we posited that mutations within the LOV2 hinge-loop, which connects the core PAS domain and the C-terminal helix, would further improve the functionality of these switches. Here, we identify hinge-loop mutations that favourably shift the dynamic range (the ratio of the on- to off-target subcellular accumulation) of the LANS and LINX photoswitches. We demonstrate the utility of these new optogenetic tools to control gene transcription and epigenetic modifications, thereby expanding the optogenetic ‘tool kit’ for the research community.

Light-induced stomatal opening stimulates CO2 uptake and transpiration in plants. Weak blue light under strong red light effectively induces stomatal opening. Blue light-dependent stomatal opening initiates light perception by phototropins, and the signal is transmitted to a plasma membrane H+-ATPase in guard cells via BLUE LIGHT SIGNALING 1 (BLUS1) kinase. However, it is unclear how BLUS1 transmits the signal to H+-ATPase. Here, we characterized BLUS1 signaling in Arabidopsis thaliana, and showed that the BLUS1 C-terminus acts as an auto-inhibitory domain and that phototropin-mediated Ser-348 phosphorylation within the domain removes auto-inhibition. C-Terminal truncation and phospho-mimic Ser-348 mutation caused H+-ATPase activation in the dark, but did not elicit stomatal opening. Unexpectedly, the plants exhibited stomatal opening under strong red light and stomatal closure under weak blue light. A decrease in intercellular CO2 concentration via red light-driven photosynthesis together with H+-ATPase activation caused stomatal opening. Furthermore, phototropins caused H+-ATPase dephosphorylation in guard cells expressing constitutive signaling variants of BLUS1 in response to blue light, possibly for fine-tuning stomatal opening. Overall, our findings provide mechanistic insights into the blue light regulation of stomatal opening.

Phototropins are photoreceptor proteins that regulate blue light-dependent biological processes for efficient photosynthesis in plants and algae. The proteins consist of a photosensory domain that responds to the ambient light and an output module that triggers cellular responses. The photosensory domain of phototropin from Chlamydomonas reinhardtii contains two conserved LOV (light-oxygen-voltage) domains with flavin chromophores. Blue light triggers the formation of a covalent cysteine-flavin adduct and upregulates the phototropin kinase activity. Little is known about the structural mechanism that leads to kinase activation and how the two LOV domains contribute to this. Here, we investigate the role of the LOV1 domain from C. reinhardtii phototropin by characterizing the structural changes occurring after blue light illumination with nano- to millisecond time-resolved X-ray solution scattering. By structurally fitting the data with atomic models generated by molecular dynamics simulations, we find that adduct formation induces a rearrangement of the hydrogen bond network from the buried chromophore to the protein surface. In particular, the change in conformation and the associated hydrogen bonding of the conserved glutamine 120 induce a global movement of the β-sheet, ultimately driving a change in the electrostatic potential on the protein surface. On the basis of the change in the electrostatics, we propose a structural model of how LOV1 and LOV2 domains interact and regulate the full-length phototropin from C. reinhardtii. This provides a rationale for how LOV photosensor proteins function and contributes to the optimal design of optogenetic tools based on LOV domains.

When exposed to fluctuating light intensity, chloroplasts move towards weak light (accumulation response), and away from strong light (avoidance response). In addition, cold treatment (5°C) induces the avoidance response even under weak-light conditions (cold-avoidance response). These three responses are mediated by the phototropin (phot), which is a blue-light photoreceptor and has also been reported to act as a thermosensory protein that perceives temperature variation. Our previous report indicated that cold-induced changes in phot biochemical activity initiate the cold-avoidance response. In this study, we further explored the induction mechanism of the cold-avoidance response in the liverwort Marchantia polymorpha and examined the relationship between changes in the amount of phot and the induction of the cold-avoidance response. The switch between the accumulation and avoidance responses occurs at a so-called 'transitional' light intensity. Our physiological experiments revealed that a cold-mediated decrease in the transitional light intensity leads to the induction of the cold-avoidance response. While artificial overexpression of phot decreased the transitional light intensity as much as cold treatment did, the amount of endogenous phot was not increased by cold treatment in wild-type M. polymorpha. Taken together, these findings show that the cold-avoidance response is initiated by a cold-mediated reduction of the transitional light intensity, independent of the amount of endogenous phot. This study provides a clue to understanding the mechanism underlying the switch in direction of chloroplast relocation in response to light and temperature.

Premise The importance of chloroplast movement for plant growth in constant, controlled light and of nonphotochemical quenching (NPQ) in variable, natural light are known. Here we concurrently investigated growth and reproduction of several Arabidopsis thaliana mutants to assess the relative importance of photoprotection via chloroplast movement and NPQ. Methods Plants were grown outdoors (natural conditions) or in a growth chamber with variable light and chilling temperatures (controlled conditions). Phenotypic growth and reproductive variables were determined at set times before maturity in wild-type (WT) and phot1, phot2, phot1phot2 (e.g., impaired chloroplast movement, stomatal conductance, leaf flattening), chup1 (impaired chloroplast movement), and npq1 (reduced NPQ) plants. Results Mutants were most adversely affected in natural conditions, with phot1phot2 and chup1 most severely impacted. These mutants bolted later and produced fewer leaves and siliques, less leaf biomass, and fewer secondary inflorescences than WT. In controlled conditions, leaf traits of these mutants were unaffected, but phot1phot2 bolted later and produced fewer secondary inflorescences and siliques than WT. For most variables, there were significant interactions between growth conditions and plant genotype. Many variables were correlated, but those relationships changed with growth conditions and genotype. Conclusions Phenotypic variables at the time of the harvest were strongly affected by growth conditions and genotype. In natural conditions, phot1phot2 and chup1 mutants were most adversely affected, demonstrating the importance of chloroplast movement. In controlled conditions, only phot1phot2 was consistently affected, also emphasizing the important, pleiotropic effects of phototropins. In both conditions, NPQ was less important.

Significance Statement This study indicates that CIPK23 is a phototropin‐interacting protein kinase that promotes blue light‐ and phototropin‐dependent stomatal opening in Arabidopsis thaliana. We propose that CIPK23 does not mediate plasma membrane H+‐ATPase activation but mediates inward‐rectifying K+ channel activation in stomatal opening., SUMMARY Phototropins (phot1 and phot2) are plant blue light receptor kinases that function to mediate phototropism, chloroplast movement, leaf flattening, and stomatal opening in Arabidopsis. Considerable progress has been made in understanding the mechanisms associated with phototropin receptor activation by light. However, the identities of phototropin signaling components are less well understood by comparison. In this study, we specifically searched for protein kinases that interact with phototropins by using an in vitro screening method (AlphaScreen) to profile interactions against an Arabidopsis protein kinase library. We found that CBL‐interacting protein kinase 23 (CIPK23) interacts with both phot1 and phot2. Although these interactions were verified by in vitro pull‐down and in vivo bimolecular fluorescence complementation assays, CIPK23 was not phosphorylated by phot1, as least in vitro. Mutants lacking CIPK23 were found to exhibit impaired stomatal opening in response to blue light but no deficits in other phototropin‐mediated responses. We further found that blue light activation of inward‐rectifying K+ (K+ in) channels was impaired in the guard cells of cipk23 mutants, whereas activation of the plasma membrane H+‐ATPase was not. The blue light activation of K+ in channels was also impaired in the mutant of BLUS1, which is one of the phototropin substrates in guard cells. We therefore conclude that CIPK23 promotes stomatal opening through activation of K+ in channels most likely in concert with BLUS1, but through a mechanism other than activation of the H+‐ATPase. The role of CIPK23 as a newly identified component of phototropin signaling in stomatal guard cells is discussed.

Key message Brachypodium distachyonis 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. Abstract 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.

Proteins are usually studied in well-defined buffer conditions, which differ substantially from those within a host cell. In some cases, the intracellular environment has an impact on the mechanism, which might be missed by in vitro experiments. Infrared difference spectroscopy previously has been applied to study the light-induced response of photoreceptors and photoenzymes in vitro Here, we established the in-cell infrared difference (ICIRD) spectroscopy in the transmission and attenuated total reflection (ATR) configuration to investigate the light-induced response of soluble proteins in living bacterial cells. ICIRD spectroscopy on the light, oxygen, or voltage (LOV) domains of the blue light receptors aureochrome and phototropin revealed a suppression of the response of specific secondary structure elements, indicating that the intracellular environment affects LOV photoreceptor mechanisms in general. Moreover, in-cell fluorescence spectroscopy disclosed that the intracellular environment slows down the recovery of the light-induced flavin adduct. Segment-resolved ICIRD spectroscopy on basic-region leucine zipper (bZIP)-LOV of aureochrome 1a from the diatom Phaeodactylum tricornutum indicated a signal progression from the LOV sensor to the bZIP effector independent of unfolding of the connecting A'alpha-helix, an observation that stood in contrast to in vitro results. This deviation was recapitulated in vitro by emulating the intracellular environment through addition of the crowding agent bovine serum albumin, but not by sucrose polymers. We conclude that ICIRD spectroscopy is a non-invasive, label-free approach for assessing conformational changes in receptors in living cells at ambient conditions. As demonstrated, these near-native responses may deviate from the mechanisms established under in vitro conditions.

Plants can sense the gravitational force. When plants perceive a change in this natural force, they tend to reorient their organs with respect to the direction of the gravity vector, i.e., the shoot stem curves up. In the present study, we performed a 4C quantitative phosphoproteomics to identify those altered protein phosphosites resulting from 150 s of reorientation of Arabidopsis plants on earth. A total of 5556 phosphopeptides were identified from the gravistimulated Arabidopsis. Quantification based on the 15N- s table i sotope l abeling i n A rabidopsis (SILIA) and computational analysis of the e x tracted i on c hromatogram (XIC) of phosphopeptides showed eight and five u nique P TM peptide a rrays (UPAs) being up- and down-regulated, respectively, by gravistimulation. Among the 13 plant reorientation-responsive protein groups, many are related to the cytoskeleton dynamic and plastid movement. Interestingly, the most gravistimulation-responsive phosphosites are three serine residues, S350, S376, and S410, of a blue light receptor Phototropin 1 (PHOT1). The immunoblots experiment confirmed that the change of gravity vector indeed affected the phosphorylation level of S410 in PHOT1. The functional role of PHOT1 in gravitropic response was further validated with gravicurvature measurement in the darkness of both the loss-of-function double mutant phot1phot2 and its complementary transgenic plant PHOT1/phot1phot2. Significance The organs of sessile organisms, plants, are able to move in response to environmental stimuli, such as gravity vector, touch, light, water, or nutrients, which is termed tropism. For instance, the bending of plant shoots to the light source is called phototropism. Since all plants growing on earth are continuously exposed to the gravitational field, plants receive the mechanical signal elicited by the gravity vector change and convert it into plant morphogenesis, growth, and development. Past studies have resulted in various hypotheses for gravisensing, but our knowledge about how the signal of gravity force is transduced in plant cells is still minimal. In the present study, we performed a SILIA-based 4C quantitative phosphoproteomics on 150-s gravistimulated Arabidopsis seedlings to explore the phosphoproteins involved in the gravitropic response. Our data demonstrated that such a short-term reorientation of Arabidopsis caused changes in phosphorylation of cytoskeleton structural proteins like Chloroplast Unusual Positioning1 (CHUP1), Patellin3 (PATL3), and Plastid Movement Impaired2 (PMI2), as well as the blue light receptor Phototropin1 (PHOT1). These results suggested that protein phosphorylation plays a crucial role in gravisignaling, and two primary tropic responses of plants, gravitropism and phototropism, may share some common components and signaling pathways. We expect that the phosphoproteins detected from this study will facilitate the subsequent molecular and cellular studies on the mechanism underlying the signal transduction in plant gravitropic response.

We studied how plants acclimated to growing conditions that included combinations of blue light (BL) and ultraviolet (UV)-A radiation, and whether their growing environment affected their photosynthetic capacity during and after a brief period of acute high light (as might happen during an under-canopy sunfleck). Arabidopsis thaliana Landsberg erecta wild-type were compared with mutants lacking functional blue light and UV photoreceptors: phototropin 1, cryptochromes (CRY1 and CRY2) and UV RESISTANT LOCUS 8 (uvr8). This was achieved using light-emitting-diode (LED) lamps in a controlled environment to create treatments with or without BL, in a split-plot design with or without UV-A radiation. We compared the accumulation of phenolic compounds under growth conditions and after exposure to 30 min of high light at the end of the experiment (46 days), and likewise measured the operational efficiency of photosystem II (phi PSII, a proxy for photosynthetic performance) and dark-adapted maximum quantum yield (F-v/F-m to assess PSII damage). Our results indicate that cryptochromes are the main photoreceptors regulating phenolic compound accumulation in response to BL and UV-A radiation, and a lack of functional cryptochromes impairs photosynthetic performance under high light. Our findings also reveal a role for UVR8 in accumulating flavonoids in response to a low UV-A dose. Interestingly, phototropin 1 partially mediated constitutive accumulation of phenolic compounds in the absence of BL. Low-irradiance BL and UV-A did not improve phi PSII and F-v/F-m upon our acute high-light treatment; however, CRYs played an important role in ameliorating high-light stress.

The geomagnetic field (GMF) is an environmental element whose instability affects plant growth and development. Despite known plant responses to GMF direction and intensity, the mechanism of magnetoreception in plants is still not known. Magnetic field variations affect many light-dependent plant processes, suggesting that the magnetoreception could require light. The objective of this work was to comprehensively investigate the influence of GMF on Arabidopsis thaliana (Col-0) photoreceptor signaling. Wild-type Arabidopsis seedlings and photoreceptor-deficient mutants (cry1cry2, phot1, phyA and phyAphyB) were exposed to near null magnetic field (NNMF, ≤40 nT) and GMF (~43 μT) under darkness and different light wavelengths. The GMF did not alter skotomorphogenic or photomorphogenic seedling development but had a significant impact on gene expression pathways downstream of cryptochrome and phytochrome photoactivation. GMF-induced changes in gene expression observed under blue light were partially associated with an alteration of cryptochrome activation. GMF impacts on phytochrome-regulated gene expression could be attributed to alterations in phytochrome protein abundance that were also dependent on the presence of cry1, cry2 and phot1. Moreover, the GMF was found to impact photomorphogenic-promoting gene expression in etiolated seedlings, indicating the existence of a light-independent magnetoreception mechanism. In conclusion, our data shows that magnetoreception alters photoreceptor signaling in Arabidopsis, but it does not necessarily depend on light.

In this study, a LOV-based fluorescent reporter (light, oxygen, or voltage-sensing domains of phototropin), termed iLOV, was adapted for Campylobacter jejuni and used to investigate promoter activity via monitoring fluorescence intensity and to study the localisation of two chemotaxis proteins. The pC46 complementation vector contains coding sequence from cj0046, a C. jejuni NCTC11168 pseudo-gene and is used to integrate cloned genes onto the C. jejuni chromosome. The pC46 vector was used to construct plasmids containing iLOV, driven by three different C. jejuni constitutive promoters and plasmids containing transcriptional fusions of the iLOV reporter and two chemoreceptors, tlp5 and tlp8. Expression from the porA promoter, pporA, produced the highest fluorescence signals compared to pfdxA (intermediate level) and pmetK (lowest level). The cellular localisation pattern of transducer-like protein (Tlp) clusters, containing Tlp5 and Tlp8, was predominately polar, with Tlp5 positioned only at one and Tlp8 at both poles. Here, we demonstrate that a iLOV fluorescent reporter can be used as a promoter probe or as a gene fusion reporter in Campylobacter spp. This is a new system uniquely placed for studying Campylobacter spp., as it combines resistance to photobleaching and functionality under microaerobic conditions.

Genetically-encoded photosensitizers produce reactive oxygen species (ROS) in response to light. Transgenic expression of fusion proteins can target the photosensitizers to specific cell regions and permit the spatial and temporal control of ROS production. These ROS-generating proteins (RGPs) are widely used for cell ablation, mutagenesis and chromophore-assisted light inactivation of target proteins. However, the species produced by RGPs are unclear due to indirect measures with confounding interpretations. Recently, the RGP mini “Singlet Oxygen Generator” (miniSOG) was engineered from Arabidopsis thaliana phototropin 2. While miniSOG produces singlet oxygen ((1)O(2)), the contribution of superoxide (O(2)(•−)) to miniSOG-generated ROS remains unclear. We measured the light-dependent O(2)(•−) production of purified miniSOG using HPLC separation of dihydroethidium (DHE) oxidation products. We demonstrate that DHE is insensitive to (1)O(2) and establish that DHE is a suitable indicator to measure O(2)(•−) production in a system that produces both (1)O(2) and O(2)(•−). We report that miniSOG produces both (1)O(2) and O(2)(•−), as can its free chromophore, flavin mononucleotide. miniSOG produced O(2)(•−) at a rate of ~4.0 μmol O(2)(•−)/min/μmol photosensitizer for an excitation fluence rate of 5.9 mW/mm(2) at 470 ± 20 nm, and the rate remained consistent across fluences (light doses). Overall, the contribution of O(2)(•−) to miniSOG phenotypes should be considered.

The combined information provided by light and temperature cues helps to optimise plant body architecture and physiology. Plants possess elaborate systems to sense and respond to these stimuli. Simultaneous perception of light and temperature by dual receptors such as phytochrome B and phototropin leads to immediate signalling convergence. Conversely, cue asynchronies initiate separate pathways and the information of the earliest cue is stored, awaiting the arrival of the later cue to control transcription. Storage mechanisms can involve changes in the activity of selected clock components or epigenetic modifications, depending on the time delay between cues (hours, days or several months). We propose a conceptual framework in which the mechanisms of integration relate to the timing of cue sensing. Fil: Casal, Jorge José. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Fisiológicas y Ecológicas Vinculadas a la Agricultura. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones Fisiológicas y Ecológicas Vinculadas a la Agricultura; Argentina Fil: Qüesta, Julia Irene. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Norwich Research Park; Reino Unido

Plants and freshwater algae devoid of flagella evolved various photomovements to optimize their photosynthetic efficiency. The filaments of Spirogyra varians exhibit complex swaying and undulating movement and form a compact mat which enables them to adjust their light exposure. Photomovement of filament fragments (1-10 cells) was analyzed using various photoreceptor and cytoskeleton inhibitors under monochromatic light. Different patterns of movement were observed under red and blue light. The filaments showed positive phototropism under blue light. Under red light, the filaments bent to undulating shape, but rapidly became unbent by a short exposure to far-red light suggesting the involvement of phytochrome in this movement. The mechanical effector for the red-light response was microtubules; the movement was inhibited effectively by the microtubule inhibitor, oryzalin. The blue-light movement was partially inhibited by the single treatment of either cytochalasin D or oryzalin, but was completely blocked when both chemicals were applied together. Phototropin-signaling inhibitors, wortmannin and LY294002, reversibly inhibited the blue-light movement. Caffeine treatment reversibly stopped the blue-light movement, while the red-light movement was not affected by calcium inhibitors. Our results suggest that the complex photomovement of S. varians is the result of a two-track control of microtubules and microfilaments signaled by the combination of phytochrome and phototropin-like receptors.

Living organisms detect changes in temperature using thermosensory molecules. However, these molecules and/or their mechanisms for sensing temperature differ among organisms. To identify thermosensory molecules in plants, we investigated chloroplast positioning in response to temperature changes and identified a blue-light photoreceptor, phototropin, that is an essential regulator of chloroplast positioning. Based on the biochemical properties of phototropin during the cellular response to light and temperature changes, we found that phototropin perceives temperature based on the temperature-dependent lifetime of the photoactivated chromophore. Our findings indicate that phototropin perceives both blue light and temperature and uses this information to arrange the chloroplasts for optimal photosynthesis. Because the photoactivated chromophore of many photoreceptors has a temperature-dependent lifetime, a similar temperature-sensing mechanism likely exists in other organisms. Thus, photoreceptors may have the potential to function as thermoreceptors.

Controlling protein degradation can be a valuable tool for posttranslational regulation of protein abundance to study complex biological systems. In the present study, we designed a light-switchable degron consisting of a light oxygen voltage (LOV) domain of Avena sativa phototropin 1 (AsLOV2) and a C-terminal degron. Our results showed that the light-switchable degron could be used for rapid and specific induction of protein degradation in HEK293 cells by light in a proteasome-dependent manner. Further studies showed that the light-switchable degron could also be utilized to mediate the degradation of secreted Gaussia princeps luciferase (GLuc), demonstrating the adaptability of the light-switchable degron in different types of protein. We suggest that the light-switchable degron offers a robust tool to control protein levels and may serves as a new and significant method for gene- and cell-based therapies.

The Arabidopsis leaf veins bundle sheath cells (BSCs) – a selective barrier to water and solutes entering the mesophyll – increase the leaf radial hydraulic conductance (Kleaf) by acidifying the xylem sap by their plasma membrane H+-ATPase, AHA2. Based on this and on the BSCs’ expression of PHOT1 and PHOT2, and the known blue-light (BL)-induced Kleaf increase, we hypothesized that, resembling the guard cells, BL perception by the BSCs’ phots activates its H+-ATPase, which, consequently, upregulates Kleaf. Indeed, under BL, the Kleaf of the knockout mutant lines phot1-5, phot2-1, phot1-5phot2-1, and aha2-4 was lower than that of the WT. BSC-only-directed complementation of phot1-5 or aha2-4 by PHOT1 or AHA2, respectively, restored the BL-induced Kleaf increase. BSC-specific silencing of PHOT1 or PHOT2 prevented such Kleaf increase. A xylem-fed kinase inhibitor (tyrphostin 9) replicated this also in WT plants. White light – ineffective in the phot1-5 mutant – acidified the xylem sap (relative to darkness) in WT and in the PHOT1-complemented phot1-5. These results, supported by BL increase of BSC protoplasts’ water permeability and cytosolic pH and their hyperpolarization by BL, identify the BSCs as a second phot-controlled water conductance element in leaves, in series with stomatal conductance. Through both, BL regulates the leaf water balance.One-Sentence summaryBlue light regulates the leaf hydraulic conductance via the bundle-sheath cells’ blue light PHOT receptors which, via an autonomous signaling pathway, activate the BSCs’ AHA2 H+-pump.

Phototropin (phot) receptor kinases play important roles in promoting plant growth by controlling light-capturing processes, such as phototropism. Phototropism is mediated through the action of NON-PHOTOTROPIC HYPOCOTYL3 (NPH3), which is dephosphorylated following phot activation. However, the functional significance of this early signaling event remains unclear. Here, we show that the onset of phototropism in dark-grown (etiolated) seedlings of Arabidopsis (Arabidopsis thaliana) and tomato (Solanum lycopersicum) is enhanced by greening (deetiolation). Red and blue light were equally effective in promoting phototropism in Arabidopsis, consistent with our observations that deetiolation by phytochrome or cryptochrome was sufficient to enhance phototropism. Increased responsiveness did not result from an enhanced sensitivity to the phytohormone auxin, nor does it involve the phot-interacting protein, ROOT PHOTOTROPISM2. Instead, deetiolated seedlings showed attenuated levels of NPH3 dephosphorylation and diminished relocalization of NPH3 from the plasma membrane during phototropism. Likewise, etiolated seedlings that lack the PHYTOCHROME-INTERACTING FACTORS (PIFs) PIF1, PIF3, PIF4, and PIF5 displayed reduced NPH3 dephosphorylation and enhanced phototropism, consistent with their constitutive photomorphogenic phenotype in darkness. Phototropic enhancement could also be achieved in etiolated seedlings by lowering the light intensity to diminish NPH3 dephosphorylation. Thus, phototropism is enhanced following deetiolation through the modulation of a phosphorylation rheostat, which in turn sustains the activity of NPH3. We propose that this dynamic mode of regulation enables young seedlings to maximize their establishment under changing light conditions, depending on their photoautotrophic capacity.

The phototropins (phots) are light-activated kinases that are critical for plant physiology and the many diverse optogenetic tools that they have inspired. Phototropins combine two blue-light-sensing Light–Oxygen–Voltage (LOV) domains (LOV1 and LOV2) and a C-terminal serine/threonine kinase domain, using the LOV domains to control the catalytic activity of the kinase. While much is known about the structure and photochemistry of the light-perceiving LOV domains, particularly in how activation of the LOV2 domain triggers the unfolding of alpha helices that communicate the light signal to the kinase domain, many questions about phot structure and mechanism remain. Recent studies have made progress addressing these questions by utilizing small-angle X-ray scattering (SAXS) and other biophysical approaches to study multidomain phots from Chlamydomonas and Arabidopsis, leading to models where the domains have an extended linear arrangement, with the regulatory LOV2 domain contacting the kinase domain N-lobe. We discuss this and other advances that have improved structural and mechanistic understanding of phot regulation in this review, along with the challenges that will have to be overcome to obtain high-resolution structural information on these exciting photoreceptors. Such information will be essential to advancing fundamental understanding of plant physiology while enabling engineering efforts at both the whole plant and molecular levels.

In 2009, the draft genome of the reference inbred line of maize (Zea mays L. spp. mays cv. B73) was published so that, using this specific corn variety, molecular analyses of physiological processes became possible. However, the morphology and developmental patterns of B73 maize, compared with that of the more frequently used hybrid varieties, have not yet been analyzed. Here, we describe organ development in seedlings of B73 maize and in those of six other hybrid cultivars, and document significant morphological as well as quantitative differences between these varieties of Z. mays. In a second set of experiments, we used etiolated seedlings of B73 maize to analyze the effect of blue light (BL) on the patterns of proteins in the tip vs. growing region of this sheath-like organ. By using two-dimensional difference gel electrophoresis (2D DIGE), coupled with tandem mass spectrometry, we detected, in the microsomal fraction of maize coleoptile tips, rapid changes in the abundance of protein spots of maize phototropin 1 and several metabolic enzymes. In the sub-apical (growing) region of the coleoptile, proteomic changes were less pronounced. These results suggest that the tip of the coleoptile of B73 maize may serve as a unique model system for dissecting BL responses in a light-sensitive plant organ of known function.