The photo-thermochemical properties and functions of Marchantia phototropin encoded by an unduplicated gene in land plant evolution.

Phototropin (phot) is a blue light photoreceptor in plants and possesses two photosensory light­oxygen-voltage (LOV1 and LOV2) domains with different photo-thermochemical properties. While liverworts contain a single copy of PHOT (e.g., MpPHOT in Marchantia polymorpha), many land plant species contain multicopy PHOT genes (e.g., AtPHOT1 and 2 in Arabidopsis thaliana) due to evolutionary gene duplication. The LOV domains of duplicated phot proteins have been studied in detail, but those of single-copy phot proteins remain to be characterized. As phot has not been duplicated in liverworts, we hypothesized that Mpphot may retain the ancestral function and photo-thermochemical properties. To learn more about the unduplicated phot proteins, we analyzed chloroplast relocation movement and the photo-thermochemical properties of LOV1 and LOV2 in Mpphot (Mpphot-LOV1 and Mpphot-LOV2, respectively). The function of Mpphot-LOV1, which induced a response to move chloroplasts to weak light (the accumulation response) in the absence of photoactive LOV2, differed from that of LOV1 of the duplicated phot proteins of A. thaliana (e.g., Atphot1-LOV1 preventing the accumulation response). On the other hand, the function of Mpphot-LOV2 was similar to that of LOV2 of the duplicated phots. The photo-thermochemical properties of Mpphot were a hybrid of those of the duplicated phots; the photochemical and thermochemical reactions of Mpphot were similar to those of the phot2- and phot1-type proteins, respectively. Our findings reveal conservation and diversification among LOV domains during phot duplication events in land plant evolution.

Cryptochrome-mediated hypocotyl phototropism was regulated antagonistically by gibberellic acid and sucrose in Arabidopsis.

Both phototropins (phot1 and phot2) and cryptochromes (cry1 and cry2) were proven as the Arabidopsis thaliana blue light receptors. Phototropins predominately function in photomovement, and cryptochromes play a role in photomorphogenesis. Although cryptochromes have been proposed to serve as positive modulators of phototropic responses, the underlying mechanism remains unknown. Here, we report that depleting sucrose from the medium or adding gibberellic acids (GAs) can partially restore the defects in phototropic curvature of the phot1 phot2 double mutants under high-intensity blue light; this restoration does not occur in phot1 phot2 cry1 cry2 quadruple mutants and nph3 (nonphototropic hypocotyl 3) mutants which were impaired phototropic response in sucrose-containing medium. These results indicate that GAs and sucrose antagonistically regulate hypocotyl phototropism in a cryptochromes dependent manner, but it showed a crosstalk with phototropin signaling on NPH3. Furthermore, cryptochromes activation by blue light inhibit GAs synthesis, thus stabilizing DELLAs to block hypocotyl growth, which result in the higher GAs content in the shade side than the lit side of hypocotyl to support the asymmetric growth of hypocotyl. Through modulation of the abundance of DELLAs by sucrose depletion or added GAs, it revealed that cryptochromes have a function in mediating phototropic curvature.

Functional characterization of GhPHOT2 in chloroplast avoidance of Gossypium hirsutum.

Chloroplast movement mediated by the plant-specific phototropin blue light photoreceptors is crucial for plants to cope with fluctuating light conditions. While chloroplasts accumulate at weak light-illuminated areas, chloroplast avoidance response mediated primarily by the phototropin2 (phot2) receptor is induced by strong light illumination. Although extensive studies have been performed on phot2-mediated chloroplast avoidance in the model plant Arabidopsis, little is known on the role of the corresponding PHOT2 orthologs in chloroplast movement in cotton. In this study, we found that chloroplast avoidance movement also occurs in the tetraploid G. hirsutum and two diploid species, G. arboreum and G. raimondii, albeit with distinct features. Further bioinformatics and genetic analysis identified the cotton PHOT2 ortholog, GhPHOT2-1, which retained a conserved role in plant chloroplast avoidance movement under strong blue light. Ghphot2-1was localized in the plasma membrane and formed aggregates after high blue light irradiation. Constitutive expression of GhPHOT2-1 restored chloroplast avoidance and accumulation response, as well as phototropism, and leaf flattening characteristics of the Arabidopsis phot2 or phot1 phot2 mutants. On the contrary, silencing of GhPHOT2-1 by virus-induced gene silencing (VIGS) disrupted high blue light-induced chloroplast avoidance movement and caused photo damage in cotton leaves. Taken together, these findings demonstrated that GhPHOT2-1 is a conserved PHOT2 ortholog in regulating chloroplast avoidance and the other aforementioned phot2-mediated responses, implicating its potential role for improving high light tolerance in cotton cultivars.

An update on aureochromes: Phylogeny - mechanism - function.

Light is important for algae, as it warrants metabolic independence via photosynthesis. In addition to the absorption of light by the photosystems, algae possess a variety of specific photoreceptors that allow the quantification of the light fluxes as well as the assessment of light qualities. About a decade ago, aureochromes have been described in the xanthophyte alga Vaucheria frigida. These proteins represent a new type of blue light photoreceptor as they possess both a light-oxygen-voltage (LOV) domain for light reception as well as a basic region leucine zipper (bZIP) domain for DNA binding, indicating that they represent light-driven transcription factors. Aureochromes so far have been detected only in a single group of algae, photosynthetic stramenopiles, but not in any other prokaryotic or eukaryotic organisms. Recent biophysical work on aureochromes in the absence and the presence of DNA revealed the mechanism of allosteric communication between the sensor and effector domains despite their unusual inversed arrangement. Different molecular models have been proposed to describe the effect of light on DNA binding. Functional characterization of mutants of the diatom Phaeodactylum tricornutum, in which the aureochrome genes have been silenced or deleted, indicate that different aureochromes may have different functions, being involved in central processes like light acclimation and regulation of the cell cycle.

REPRESSOR OF ULTRAVIOLET-B PHOTOMORPHOGENESIS function allows efficient phototropin mediated ultraviolet-B phototropism in etiolated seedlings.

Ultraviolet B (UV-B) light is a part of the solar radiation which has significant effects on plant morphology, even at low doses. In Arabidopsis, many of these morphological changes have been attributed to a specific UV-B receptor, UV resistance locus 8 (UVR8). Recent findings showed that next to phototropin regulated phototropism, UVR8 mediated signaling is able of inducing directional bending towards UV-B light in etiolated seedlings of Arabidopsis, in a phototropin independent manner. In this study, kinetic analysis of phototropic bending was used to evaluate the relative contribution of each of these pathways in UV-B mediated phototropism. Diminishing UV-B light intensity favors the importance of phototropins. Molecular and genetic analyses suggest that UV-B is capable of inducing phototropin signaling relying on phototropin kinase activity and regulation of NPH3. Moreover, enhanced UVR8 responses in the UV-B hypersensitive rup1rup2 mutants interferes with the fast phototropin mediated phototropism. Together the data suggest that phototropins are the most important receptors for UV-B induced phototropism in etiolated seedlings, and a RUP mediated negative feedback pathway prevents UVR8 signaling to interfere with the phototropin dependent response.

Fine tuning chloroplast movements through physical interactions between phototropins.

Phototropins are plant photoreceptors which regulate numerous responses to blue light, including chloroplast relocation. Weak blue light induces chloroplast accumulation, whereas strong light leads to an avoidance response. Two Arabidopsis phototropins are characterized by different light sensitivities. Under continuous light, both can elicit chloroplast accumulation, but the avoidance response is controlled solely by phot2. As well as continuous light, brief light pulses also induce chloroplast displacements. Pulses of 0.1s and 0.2s of fluence rate saturating the avoidance response lead to transient chloroplast accumulation. Longer pulses (up to 20s) trigger a biphasic response, namely transient avoidance followed by transient accumulation. This work presents a detailed study of transient chloroplast responses in Arabidopsis. Phototropin mutants display altered chloroplast movements as compared with the wild type: phot1 is characterized by weaker responses, while phot2 exhibits enhanced chloroplast accumulation, especially after 0.1s and 0.2s pulses. To determine the cause of these differences, the abundance and phosphorylation levels of both phototropins, as well as the interactions between phototropin molecules are examined. The formation of phototropin homo- and heterocomplexes is the most plausible explanation of the observed phenomena. The physiological consequences of this interplay are discussed, suggesting the universal character of this mechanism that fine-tunes plant reactions to blue light. Additionally, responses in mutants of different protein phosphatase 2A subunits are examined to assess the role of protein phosphorylation in signaling of chloroplast movements.

A subtracted cDNA library identifies genes up-regulated during PHOT1-mediated early step of de-etiolation in tomato (Solanum lycopersicum L.).

BACKGROUND: De-etiolation is the switch from skoto- to photomorphogenesis, enabling the heterotrophic etiolated seedling to develop into an autotrophic plant. Upon exposure to blue light (BL), reduction of hypocotyl growth rate occurs in two phases: a rapid inhibition mediated by phototropin 1 (PHOT1) within the first 30-40 min of illumination, followed by the cryptochrome 1 (CRY1)-controlled establishment of the steady-state growth rate. Although some information is available for CRY1-mediated de-etiolation, less attention has been given to the PHOT1 phase of de-etiolation. RESULTS: We generated a subtracted cDNA library using the suppression subtractive hybridization method to investigate the molecular mechanisms of BL-induced de-etiolation in tomato (Solanum lycopersicum L.), an economically important crop. We focused our interest on the first 30 min following the exposure to BL when PHOT1 is required to induce the process. Our library generated 152 expressed sequence tags that were found to be rapidly accumulated upon exposure to BL and consequently potentially regulated by PHOT1. Annotation revealed that biological functions such as modification of chromatin structure, cell wall modification, and transcription/translation comprise an important part of events contributing to the establishment of photomorphogenesis in young tomato seedlings. Our conclusions based on bioinformatics data were supported by qRT-PCR analyses the specific investigation of V-H(+)-ATPase during de-etiolation in tomato. CONCLUSIONS: Our study provides the first report dealing with understanding the PHOT1-mediated phase of de-etiolation. Using subtractive cDNA library, we were able to identify important regulatory mechanisms. The profound induction of transcription/translation, as well as modification of chromatin structure, is relevant in regard to the fact that the entry into photomorphogenesis is based on a deep reprograming of the cell. Also, we postulated that BL restrains the cell expansion by the rapid modification of the cell wall.

Phototropins do not alter accumulation of evening-phased circadian transcripts under blue light.

The circadian system induces rhythmic variation in a suite of biochemical and physiological processes that serve to optimise plant growth in diel cycles. To be of greatest utility, these rhythmic behaviors are coordinated with regular environmental changes such as the rising and setting of the sun. Photoreceptors, along with metabolites produced during photosynthesis, act to synchronise the internal timing mechanism with lighting cues. We have recently shown that phototropins help maintain robust rhythms of photosynthetic operating efficiency (ϕPSII or Fq'/Fm') under blue light, although rhythmic accumulation of morning-phased circadian transcripts in the nucleus was unaffected. Here we report that evening-phased nuclear clock transcripts were also unaffected. We also observe that rhythms of nuclear clock transcript accumulation are maintained in phototropin mutant plants under a fluctuating lighting regime that induced a loss of Fq'/Fm' rhythms.

Lipid anchoring of Arabidopsis phototropin 1 to assess the functional significance of receptor internalization: should I stay or should I go?

The phototropin 1 (phot1) blue light receptor mediates a number of adaptive responses, including phototropism, that generally serve to optimize photosynthetic capacity. Phot1 is a plasma membrane-associated protein, but upon irradiation, a fraction is internalized into the cytoplasm. Although this phenomenon has been reported for more than a decade, its biological significance remains elusive. Here, we use a genetic approach to revisit the prevalent hypotheses regarding the functional importance of receptor internalization. Transgenic plants expressing lipidated versions of phot1 that are permanently anchored to the plasma membrane were used to analyse the effect of internalization on receptor turnover, phototropism and other phot1-mediated responses. Myristoylation and farnesylation effectively prevented phot1 internalization. Both modified photoreceptors were found to be fully functional in Arabidopsis, rescuing phototropism and all other phot1-mediated responses tested. Light-mediated phot1 turnover occurred as in the native receptor. Furthermore, our work does not provide any evidence of a role of phot1 internalization in the attenuation of receptor signalling during phototropism. Our results demonstrate that phot1 signalling is initiated at the plasma membrane. They furthermore indicate that release of phot1 into the cytosol is not linked to receptor turnover or desensitization.

Distinct palisade tissue development processes promoted by leaf autonomous signalling and long-distance signalling in Arabidopsis thaliana.

Plants develop palisade tissue consisting of cylindrical mesophyll cells located at the adaxial side of leaves in response to high light. To understand high light signalling in palisade tissue development, we investigated leaf autonomous and long-distance signal responses of palisade tissue development using Arabidopsis thaliana. Illumination of a developing leaf with high light induced cell height elongation, whereas illumination of mature leaves with high light increased cell density and suppressed cell width expansion in palisade tissue of new leaves. Examination using phototropin1 phototropin2 showed that blue light signalling mediated by phototropins was involved in cell height elongation of the leaf autonomous response rather than the cell density increase induced by long-distance signalling. Hydrogen peroxide treatment induced cylindrical palisade tissue cell formation in both a leaf autonomous and long-distance manner, suggesting involvement of oxidative signals. Although constitutive expression of transcription factors involved in systemic-acquired acclimation to excess light, ZAT10 and ZAT12, induced cylindrical palisade tissue cell formation, knockout of these genes did not affect cylindrical palisade tissue cell formation. We conclude that two distinct signalling pathways - leaf autonomous signalling mostly dependent on blue light signalling and long-distance signalling from mature leaves that sense high light and oxidative stress - control palisade tissue development in A. thaliana.

Phototropism: growing towards an understanding of plant movement.

Phototropism, or the differential cell elongation exhibited by a plant organ in response to directional blue light, provides the plant with a means to optimize photosynthetic light capture in the aerial portion and water and nutrient acquisition in the roots. Tremendous advances have been made in our understanding of the molecular, biochemical, and cellular bases of phototropism in recent years. Six photoreceptors and their associated signaling pathways have been linked to phototropic responses under various conditions. Primary detection of directional light occurs at the plasma membrane, whereas secondary modulatory photoreception occurs in the cytoplasm and nucleus. Intracellular responses to light cues are processed to regulate cell-to-cell movement of auxin to allow establishment of a trans-organ gradient of the hormone. Photosignaling also impinges on the transcriptional regulation response established as a result of changes in local auxin concentrations. Three additional phytohormone signaling pathways have also been shown to influence phototropic responsiveness, and these pathways are influenced by the photoreceptor signaling as well. Here, we will discuss this complex dance of intra- and intercellular responses that are regulated by these many systems to give rise to a rapid and robust adaptation response observed as organ bending.

Sustained accurate recording of intracellular acidification in living tissues with a photo-controllable bioluminescent protein.

Regulation of an intracellular acidic environment plays a pivotal role in biological processes and functions. However, spatiotemporal analysis of the acidification in complex tissues of living subjects persists as an important challenge. We developed a photo-inactivatable bioluminescent indicator, based on a combination of luciferase-fragment complementation and a photoreaction of a light, oxygen, and voltage domain from Avena sativa Phototropin1 (LOV2), to visualize temporally dynamic acidification in living tissue samples. Bioluminescence of the indicator diminished upon light irradiation and it recovered gradually in the dark state thereafter. The recovery rate was remarkably sensitive to pH changes but unsusceptible to fluctuation of luciferin or ATP concentrations. Bioluminescence imaging, taken as an index of the recovery rates, enabled long-time recording of acidification in apoptotic and autophagous processes in a cell population and an ischemic condition in living mice. This technology using the indicator is widely applicable to sense organelle-specific acidic changes in target biological tissues.

Evolution of three LOV blue light receptor families in green plants and photosynthetic stramenopiles: phototropin, ZTL/FKF1/LKP2 and aureochrome.

Many organisms, including bacteria, fungi, animal, plants and algae, utilize blue light to adapt to a fluctuating light environment. Plants and algae, and photosynthetic stramenopiles in particular, require light energy for photosynthesis and have thus evolved a range of sophisticated light-sensing systems to utilize light information efficiently for growth, development and physiological responses. LOV (light, oxygen or voltage) domain photoreceptors are widely distributed among prokaryotic and eukaryotic organisms, and the number of specific LOV photoreceptors are increased in certain taxa. In this review, we summarize the molecular basis and physiological functions of three different families of LOV blue light receptors specific to green plants and photosynthetic stramenopiles: phototropin, ZEITLUPE/FLAVIN-BINDING, KELCH REPEAT, F-BOX 1/LOV KELCH PROTEIN 2 (ZTL/FKF1/LKP2) and aureochrome.

A wandering pathway in plant biology: from wildflowers to phototropins to bacterial virulence.

The author describes the somewhat convoluted pathway he followed from amateur taxonomy of Minnesota wildflowers to identification of the phototropin family of blue-light receptors. He also mentions individuals who were important in moving his career first into plant taxonomy, then plant development, and finally plant photobiology (and out of music). He emphasizes the many twists and turns a research career can take, including a few that lead to blind ends. He also emphasizes the oscillatory nature of his career-back and forth between the Atlantic and Pacific oceans (with occasional forays to Freiburg, Germany) and back and forth between red-light receptors and blue-light receptors. There is a short intermission in which he describes his longtime relationship with California's Henry W. Coe State Park. Finally, he relates how he followed the unlikely pathway from plant blue-light receptors to a blue-light receptor required to maximize virulence of a bacterial animal pathogen.

Leaf positioning of Arabidopsis in response to blue light.

Appropriate leaf positioning is essential for optimizing photosynthesis and plant growth. However, it has not been elucidated how green leaves reach and maintain their position for capturing light. We show here the regulation of leaf positioning under blue light stimuli. When 1-week-old Arabidopsis seedlings grown under white light were transferred to red light (25 micromol m(-2) s(-1)) for 5 d, new petioles that appeared were almost horizontal and their leaves were curled and slanted downward. However, when a weak blue light from above (0.1 micromol m(-2) s(-1)) was superimposed on red light, the new petioles grew obliquely upward and the leaves were flat and horizontal. The leaf positioning required both phototropin1 (phot1) and nonphototropic hypocotyl 3 (NPH3), and resulted in enhanced plant growth. In an nph3 mutant, neither optimal leaf positioning nor leaf flattening by blue light was found, and blue light-induced growth enhancement was drastically reduced. When blue light was increased from 0.1 to 5 micromol m(-2) s(-1), normal leaf positioning and leaf flattening were induced in both phot1 and nph3 mutants, suggesting that phot2 signaling became functional and that the signaling was independent of phot1 and NPH3 in these responses. When plants were irradiated with blue light (0.1 micromol m(-2) s(-1)) from the side and red light from above, the new leaves became oriented toward the source of blue light. When we transferred these plants to both blue light and red light from above, the leaf surface changed its orientation to the new blue light source within a few hours, whereas the petioles initially were unchanged but then gradually rotated, suggesting the plasticity of leaf positioning in response to blue light. We showed the tissue expression of NPH3 and its plasma membrane localization via the coiled-coil domain and the C-terminal region. We conclude that NPH3-mediated phototropin signaling optimizes the efficiency of light perception by inducing both optimal leaf positioning and leaf flattening, and enhances plant growth.

Use of confocal laser as light source reveals stomata-autonomous function.

In most terrestrial plants, stomata open during the day to maximize the update of CO(2) for photosynthesis, but they close at night to minimize water loss. Blue light, among several environmental factors, controls this process. Stomata response to diverse stimuli seems to be dictated by the behaviour of neighbour stomata creating leaf areas of coordinated response. Here individual stomata of Arabidopsis leaves were illuminated with a short blue-light pulse by focusing a confocal argon laser. Beautifully, the illuminated stomata open their pores, whereas their dark-adapted neighbours unexpectedly experience no change. This induction of individual stomata opening by low fluence rates of blue light was disrupted in the phototropin1 phototropin2 (phot1 phot2) double mutant, which exhibits insensitivity of stomatal movements in blue-illuminated epidermal strips. The irradiation of all epidermal cells making direct contact with a given stoma in both wild type and phot1 phot2 plants does not trigger its movement. These results unravel the stoma autonomous function in the blue light response and illuminate the implication of PHOT1 and/or PHOT2 in such response. The micro spatial heterogeneity that solar blue light suffers in partially shaded leaves under natural conditions highlights the physiological significance of the autonomous stomatal behaviour.