G-proteins in etiolated Avena seedlings. Possible phytochrome regulation

The molecular mechanism of light signal transduction in plants mediated by the photosensor phytochrome is not well understood. The possibility that phytochrome initiates the signal transduction chain by modulating a G-protein-like receptor is examined in the present work. Etiolated Avena seedlings contain G-proteins as examined in terms of the binding of GTP as well as by cross-reaction with mammalian G-protein antibodies. The binding of GTP was regulated in vivo by red/far-red light. The possible involvement of G-proteins in the phytochrome-mediated signal transduction in etiolated Avena seedlings has been implicated from the study of the light regulated expression of the Cab and phy genes.     

Altered Phytochrome Regulation of Greening in an aurea Mutant of Tomato

A brief pulse of red light accelerates chlorophyll accumulation upon subsequent transfer of dark-grown tomato (Lycopersicon esculentum) seedlings to continuous white light. Such potentiation of greening was compared in wild type and an aurea mutant W616. This mutant has been the subject of recent studies of phytochrome phototransduction; its dark-grown seedlings are deficient in phytochrome, and light-grown plants have yellow-green leaves. The rate of greening was slower in the mutant, but the extent (relative to the dark control) of potentiation by the red pulse was similar to that in the wild type. In the wild type, the fluence-response curve for potentiation of greening indicates substantial components in the VLF (very low fluence) and LF (low fluence) ranges. Far-red light could only partially reverse the effect of red. In the aurea mutant, only red light in the LF range was effective, and the effect of red was completely reversed by far-red light. When grown in total darkness, aurea seedlings are also deficient in photoconvertible PChl(ide). Upon transfer to white light, the aurea mutant was defective in both the abundance and light regulation of the light-harvesting chlorophyll a/b binding polypeptide(s) [LHC(II)]. The results are consistent with the VLF response in greening being mediated by phytochrome. Furthermore, the data support the hypothesis that light modulates LHC(II) levels through its control of the synthesis of both chlorophyll and its LHC(II) apoproteins. Some, but not all, aspects of the aurea phenotype can be accounted for by the deficiency in photoreception by phytochrome.     

Overexpression of the Heterotrimeric G-Protein α-Subunit Enhances Phytochrome-Mediated Inhibition of Hypocotyl Elongation in Arabidopsis

Plant heterotrimeric G-proteins have been implicated in a number of signaling processes. However, most of these studies are based on biochemical or pharmacological approaches. To examine the role of heterotrimeric G-proteins in plant development, we generated transgenic Arabidopsis expressing the Galpha subunit of the heterotrimeric G-protein under the control of a glucocorticoid-inducible promoter. With the conditional overexpression of either the wild type or a constitutively active version of Arabidopsis Galpha, transgenic seedlings exhibited a hypersensitive response to light. This enhanced light sensitivity was more exaggerated in a relatively lower intensity of light and was observed in white light as well as far-red, red, and blue light conditions. The enhanced responses in far-red and red light required functional phytochrome A and phytochrome B, respectively. Furthermore, the response to far-red light depended on functional FHY1 but not on FIN219 and FHY3. This dependence on FHY1 indicates that the Arabidopsis Galpha protein may act only on a discrete branch of the phytochrome A signaling pathway. Thus, our results support the involvement of a heterotrimeric G-protein in the light regulation of Arabidopsis seedling development.     

Polyclonal antibodies raised to phycocyanins contain components specific for the red-absorbing form of phytochrome

Polyclonal antibodies raised in rabbits to a mixture of sodium-dodecyl-sulphate-denatured C- and allo-phycocyanin, isolated from Anabaena cylindrica, cross-react with 124-kilodalton (kDa) phytochrome from etiolated oats, in enzyme-linked immunosorbent assays and on Western blots. The component(s) of the anti-phycocyanin serum that cross-reacts with phytochrome appears to be specific for the red-absorbing form of phytochrome (Pr). These antibodies can be detached from Pr by irradiation with red light, and thus show photoreversible binding. This property has been used to immunopurify the anti-phytochrome component from the antiserum using red light as the eluting agent. Competition assays and epitope-mapping studies indicate that the anti-phytochrome component may bind to a site located between 6 and 10 kDa from the amino-terminus of etiolated oat phytochrome.Abbreviations ELISA enzyme-linked immunosorbent assay - kDa kilodaton - FR far-red light - Pfr far-red-light-absorbing form of phytochrome - Pr red-light-absorbing form of phytochrome - R red light - SDS sodium dodecyl sulphate     

Involvement of phytochrome and a blue light photoreceptor in UV-B induced flavonoid synthesis in parsley (Petroselinum hortense Hoffm.) cell suspension cultures

Flavonoid synthesis in cell suspension cultures of parsley (Petroselinum hortense Hoffm.) occurs only after irradiation with ultraviolet light (UV), mainly from the UV-B (280–320 nm) spectral range. However, it is also controlled by phytochrome. A P_fr/P_tot ratio of approximately 20% is sufficient for a maximum phytochrome response as induced by pulse irradiation. Continuous red and far red light, as well as blue light, given after UV, are more effective than pulse irradiations. The response to blue light is considerably greater than that to red and far red light. Continuous red and blue light treatments can be substituted for by multiple pulses and can thus probably be ascribed to a multible induction effect. Continuous irradiations with red, far red and blue light also increase the UV-induced flavonoid synthesis if given before UV. The data indicate that besides phytochrome a separate blue light photoreceptor is involved in the regulation of the UV-induced flavonoid synthesis. This blue light receptor seems to require the presence of P_fr in order to be fully effective.Abbreviations HIR high irradiance response - P_fr far red absorhing form of phytochrome - P_tet total phytochrome - UV ultraviolet light     

The involvement of phytochrome in controlling chlorophyll and 5-aminolevulinate formation in a gymnosperm seedling (Pinus sylvestris)

Chlorophyll a (Chl a) accumulation in the cotyledons of Scots pine seedlings (Pinus sylvestris L.) is much higher in the light than in darkness where it ceases 6 days after germination. When these darkgrown seedlings are treated with continuous white light (3,500 lx) a 3 h lag phase appears before Chl a accumulation is resumed. The lag phase can be eliminated by pretreating the seedlings with 7 h of weak red light (0.14 Wm^-2) or with 14 red light pulses separated by relatively short dark periods (<100 min). The effect of 15s red light pulses can be fully reversed by 1 min far-red light pulses. This reversibility is lost within 2 min. In addition, the amount of Chl a formed within 27 h of continuous red light is considerably reduced by the simultaneous application of far-red (RG 9) light. It is concluded that phytochrome (P_fr) is required not only for the elimination of the lagphase but also to maintain a high rate of Chl a accumulation in continuous light. Since accumulation of 5-aminolevulinate (ALA) responds in the same manner as Chl a accumulation to a red light pretreatment it is further concluded that ALA formation is the point where phytochrome regulates Chl biosynthesis in continuous light. No correlation has been found between ALA and Chl a formation in darkness. This indicates that in a darkgrown pine seedling ALA formation is not rate limiting for Chl a accumulation.Abbreviations Chl chlorophyll(ide) - PChl protochlorophyll(ide) - ALA 5-aminolevulinate - P_r the red absorbing form of phytochrome - P_fr the far-red absorbing form of phytochrome - P_tot total phytochrome ([P_r]+[P_fr])     

Visualization of a rapid,red/far-red light-dependent reaction by centrifuge microscopy

Summary Using a centrifuge microscope with stroboscopic illumination, we examined the effects of light irradiation on the passive movement of chloroplasts in dark-adapted mesophyll cells ofVallisneria gigantea. While irradiation with red light accelerates the passive gliding of chloroplasts produced by centrifugal force, irradiation with far-red light negates this effect. Irradiation with blue light does not accelerate the passive gliding, while red light is completely effective even in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, an inhibitor of photosynthesis. An apparently active movement of chloroplasts can be induced by irradiation with red or blue light only in the presence of the far-red light-absorbing form of phytochrome. The significance of the reaction in the light with respect to the regulation of cytoplasmic streaming is discussed.Abbreviations APW artificial pond water - CMS centrifuge microscope of the stroboscopic type - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - Pfr phytochrome, far-red light-absorbing form - Pr phytochrome, red light-absorbing form     

Intracellular localisation of phytochrome in oat coleoptiles by electron microscopy

The intracellular localisation of phytochrome in oat (Avena sativa L. cv. Garry Oat) coleoptiles was analysed by electron microscopy. Serial ultrathin sections of resin-embedded material were indirectly immunolabeled with polyclonal antibodies against phytochrome together with a gold-coupled second antibody. The limits of detectability of sequestered areas of phytochrome (SAPs) were analysed as a function of light pretreatments and amounts of the far-red absorbing form of phytochrome (Pfr) established. In 5-d-old dark-grownAvena coleoptiles SAPs were not detectable if less than 13 units of Pfr — compared with 100 units total phytochrome of 5-d-old dark-grown seedlings — were established by a red light pulse. In other sets of experiments, seedlings were preirradiated either with a non-saturating red light pulse to allow destruction to occur or with a saturating red followed by a far-red light pulse to induce first SAP formation and then its disaggregation. These preirradiations resulted in an increase of the limit of detectability of SAP formation after a second red light pulse to 38–41 and 19–23 units Pfr, respectively. We conclude that with respect to Pfr-induced SAP formation an adaptation process exists and that our data indicate that SAP formation is not a simple self-aggregation of newly formed Pfr.Abbreviations FR far-red light - Pfr, Pr far-red-absorbing and red-absorbing forms of phytochrome, respectively - Plot total phytochrome (Pfr + Pr) - R red light - SAP sequestered areas of phytochrome This work was supported by Deutsche Forschungsgemeinschaft (SFB 206). The competent technical assistance of Karin Fischer is gratefully acknowledged.     

Action spectrum and characteristics of the light activated disappearance of phytochrome in oat seedlings

The action spectrum for the light-activated destruction of phytochrome in etiolated Avena seedlings has been determined. There are 2 broad maxima, one between 380 and 440 mμ, the other between 600 and 700 mμ. peaking at about 660 mμ. On an incident energy basis, the red region of the spectrum is more efficient than the blue by about one order of magnitude in activating phytochrome disappearance. Both the red absorbing as well as the far-red absorbing forms of phytochrome are destroyed after exposure of Avena seedling to either red or blue light.

From the action spectrum and photoreversibility of pigment loss, we conclude that phytochrome acts as a photoreceptor for the photoactivation of its metabolically-based destruction. We suggest that another pigment might also be associated with the disappearance of phytochrome in oat seedlings exposed to blue light.

     

The kinetics of type 1 phytochrome in green,light-grown wheat (Triticum aestivum L.)

The kinetics of type 1 phytochrome were investigated in green, light-grown wheat. Phytochrome was measured by a quantitative sandwich enzyme-linked immunosorbent assay using monoclonal antibodies. The assay was capable of detecting down to 150 pg of phytochrome. In red light, rapid first-order destruction of the far-red-light-absorbing form of phytochrome (Pfr) with a half-life of 15 min was observed. Following white light terminated by red, phytochrome synthesis was delayed in darkness by about 15 h compared to plants given a terminal far-red treatment. Synthesis of the red-light-absorbing form of phytochrome (Pr) was zero-order in these experiments. Phytochrome synthesis in far-red light was approximately equal to synthesis in darkness in wheat although net destruction occurred in light-grown Avena sativa tissues in continuous far-red light, as has been reported for other monocotyledons. In wheat, destruction of Pfr apparently did not occur below a certain threshold level of Pfr or Pfr/total phytochrome. These results are consistent with an involvement of type 1 phytochrome in the photoperiodic control of flowering in wheat and other long-day plants.Abbreviations ELISA enzyme-linked immunosorbent assay - FR far-red light - HIR high-irradiance response - Pfr farred-light-absorbing form of phytochrome - Pr red-light-absorbing form of phytochrome - Ptot total phytochrome (Pr + Pfr) - R red light The authors wish to thank Prof. Daphne Vince-Prue (University of Reading) for many helpful discussions regarding this work. Hugh Carr-Smith was supported by a Science and Engineering Research Council studentship and Chris Plumpton by an Agricultural and Food Research Council (AFRC) studentship. B. Thomas and G. Butcher were supported by the AFRC.     

The protein phosphatase 7 regulates phytochrome signaling in Arabidopsis

The psi2 mutant of Arabidopsis displays amplification of the responses controlled by the red/far red light photoreceptors phytochrome A (phyA) and phytochrome B (phyB) but no apparent defect in blue light perception. We found that loss-of-function alleles of the protein phosphatase 7 (AtPP7) are responsible for the light hypersensitivity in psi2 demonstrating that AtPP7 controls the levels of phytochrome signaling. Plants expressing reduced levels of AtPP7 mRNA display reduced blue-light induced cryptochrome signaling but no noticeable deficiency in phytochrome signaling. Our genetic analysis suggests that phytochrome signaling is enhanced in the AtPP7 loss of function alleles, including in blue light, which masks the reduced cryptochrome signaling. AtPP7 has been found to interact both in yeast and in planta assays with nucleotide-diphosphate kinase 2 (NDPK2), a positive regulator of phytochrome signals. Analysis of ndpk2-psi2 double mutants suggests that NDPK2 plays a critical role in the AtPP7 regulation of the phytochrome pathway and identifies NDPK2 as an upstream element involved in the modulation of the salicylic acid (SA)-dependent defense pathway by light. Thus, cryptochrome- and phytochrome-specific light signals synchronously control their relative contribution to the regulation of plant development. Interestingly, PP7 and NDPK are also components of animal light signaling systems.     

Far-Red Reversal of Red Light Effect during Long-Night Induction of Potato (Solanum tuberosum L.) Tuberization

The hypothesis that phytochrome is involved in the regulation of potato (Solanum tuberosum L.) tuberization was tested. When 5 minutes of red light were given in the middle of the 16-hour dark period to which whole plants were exposed daily for 14 days before making cuttings, the percentage of tuberization on cuttings decreased. The effect of red light was significantly reversed by 2 minutes of far-red light given immediately after the red in each of two separate experiments. This supports the hypothesis that phytochrome is at least indirectly involved.     

Light-induced linear dichroism in photoreversibly photochromic sensor pigments. – V. Reinterpretation of the experiments on in vivo action dichroism of phytochrome

Experiments by several authors on the effects of polarized light on phytochromemediated responses in fern gametophytes and in the green alga Mougeotia have earlier been interpreted as showing that the transition moment of phytochrome in the P_r form is parallel to the plasmalemma, but perpendicular to the plasmalemma for the P_fr form of phytochrome. It is now shown that the experimental results can be interpreted differently, and that they are also consistent with a chromophore rotation of about 30° (instead of 90°), as found for immobilized phytochrome molecules in vitro. Thus there is no evidence for a rotation of the whole phytochrome protein. For the gametophyte of Adiantum it is calculated that the P_r transition moment is inclined 17° to the plasmalemma, and the P_fr transition moment ca 50°, corresponding to an in vivo chromophore rotation of ca 33°; however, these values are very approximate.     

Succinate dehydrogenase in Arabidopsis thaliana is regulated by light via phytochrome A

The effect of light on succinate dehydrogenase (SDH) activity and mRNA content was studied in Arabidopsis thaliana plants. The transition from darkness to light caused a short transient increase in the SDH activity followed by a decrease to a half of the original activity. The white or red light were found to be down-regulating factors for the mRNA content of the sdh1-2 and sdh2-3 genes and SDH catalytic activity both in A. thaliana wild-type plants and in the mutant deficient in the phytochrome B gene, but not in the mutant deficient in the phytochrome A gene, while the far-red light of 730 nm reversed the red light effect. It is concluded that phytochrome A participates in the regulation of mitochondrial respiration through effect on SDH expression.     

Analysis of light-controlled accumulation of carotenoids in mustard (Sinapis alba L.) seedlings

Carotenoid accumulation in the cotyledons of the mustard seedling (Sinapis alba L.) is controlled by light. Besides the stimulatory function of phytochrome in carotenogenesis the experiments reveal the significance of chlorophyll accumulation for the accumulation of larger amounts of acrotenoids. A specific blue light effect was not found. The data suggest that light exerts its control over carotenoid biogenesis through two separate mechanisms: A phytochrome regulation of enzyme levels before a postulated pool of free carotenoids, and a regulation by chlorophyll draining the pool by complex-formation.Abbreviations Chl chlorophyll(s) - PChl protochlorophyll(ide) - HIR high irradiance reaction (of phytochrome) - P_fr far-red absorbing, physiologically active form of phytochrome - P_r red absorbing, physiologically inactive form of phytochrome - P_fof total phytochrome, i.e. [P_r]+[P_fr] - [P_fr]/[P_fof], wavelength dependent photoequilibrium of the phytochrome system - red red light - fr far-red light     

Osmotic adjustment in Spirulina platensis

Unilateral irradiation of maize (Zea mays L.) seedlings results in a fluence-rate gradient, and hence below saturation, a gradient of the far-red-absorbing form of phytochrome (Pfr). The Pfr-gradients established by blue, red and far-red light were spectrophotometrically measured in the mesocotyl. Based on these Pfr-gradients and the fluence-response curves of phytochrome photoconversion the fluence-rate gradients were calculated. The fluence-rate gradient in the blue (460 nm) was steeper than that in the red (665 nm), which in turn was steeper than that in the far-red light (725 nm). The fluence-rate ratios front to rear were 1:0.06 (460 nm), 1:0.2 (665 nm), and 1:0.33 (725 nm). The assumption that phytochrome-mediated phototropism of maize mesocotyls is caused by local phytochrome-mediated growth inhibition was tested in the following manner. Firstly, the Pfr response curve for growth inhibition was calculated; these calculations were based on measurements of Pfr-gradients and data from red-light-induced phototropism. Secondly, the Pfr response curve for growth inhibition was used as a basis for calculating fluence-response curves for blue-and far-red-light-induced phototropism. Finally, these calculated results were compared with experimental data. It was concluded that the threshold for phytochrome-mediated phototropism of maize mesocotyls reflects the apparent photoconversion cross section of phytochrome whereas the maximal inducable curvature depends on the steepness of the light (Pfr) gradient across the mesocotyl.Abbreviations Pfr far-red-absorbing form of phytochrome - Ptot total phytochrome - Fr far-red light     

Effects of light on phytochrome in cauliflower curd

The effect of light on the phytochrome content of cauliflower (Brassica oleracea (L.) var. botrytis) curd was studied using in vivo spectrophotometry. It was found that light caused a rapid increase in phytochrome level whereas transfer to darkness caused a rapid loss, regardless of the amount of phytochrome initially present in the far red absorbing form. The amount of phytochrome detectable during continuous irradiation appears to be related to the photoequilibrium , and is thus controlled by phytochrome itself.Abbreviation Pr and Pfr red and far red absorbing forms of phytochrome, respectively     

Reorientation of microtubules at the outer epidermal wall of maize coleoptiles by phytochrome,blue-light photoreceptor,and auxin

Summary The effects of red and blue light on the orientation of cortical microtubules (MTs) underneath the outer epidermal wall of maize (Zea mays L.) coleoptiles were investigated with immunofluorescent techniques. The epidermal cells of dark-grown coleoptiles demonstrated an irregular pattern of regions of parallel MTs with a random distribution of orientations. This pattern could be changed into a uniformly transverse MT alignment with respect to the long cell axis by 1 h of irradiation with red light. This response was transient as the MTs spontaneously shifted into a longitudinal orientation after 1–2 h of continued irradiation. Induction/reversion experiments with short red and far-red light pulses demonstrated the involvement of phytochrome in this response. In contrast to red light, irradiation with blue light induced a stable longitudinal MT alignment which was established within 10 min. The blue-light response could not be affected by subsequent irradiations with red or far-red light indicating the involvement of a separate blue-light photoreceptor which antagonizes the effect of phytochrome. In mixed light treatments with red and blue light, the blue-light photoreceptor always dominated over phytochrome which exhibited an apparently less stable influence on MT orientation. Long-term irradiations with red or blue light up to 6 h did not reveal any rhythmic changes of MT orientation that could be related to the rhythmicity of helicoidal cell-wall structure. Subapical segments isolated from dark-grown coleoptiles maintained a longitudinal MT arrangement even in red light indicating that the responsiveness to phytochrome was lost upon isolation. Conversely auxin induced a transverse MT arrangement in isolated segments even in blue light, indicating that the responsiveness to blue-light photoreceptor was eliminated by the hormone. These complex interactions are discussed in the context of current hypotheses on the functional significance of MT reorientations for cell development.Abbreviations MT cortical microtubule - P_r, P_fr red and far-red absorbing form of phytochrome     

Reversible phytochrome regulation influenced the severity of ozone-induced visible foliar injuries in Trifolium subterraneum L.

In Trifolium subterraneum, oxidative stress caused by ozone has been shown to result in more severe visible foliar injuries when plants were kept in dim broadband white light during the night (i.e. a long photoperiod) compared to darkness during the night (a short photoperiod). As phytochrome signalling is involved in photoperiod sensing, the effect of night-time red and far-red illumination on the ozone-induced response was studied. T. subterraneum plants were treated with ozone enriched air (70?ppb) for either 1?h for a single day or 6?h for three consecutive days. After the first ozone exposure, plants were separated into six night-time light regimes during the two subsequent nights (10?h?day, 14?h night): (1) darkness, (2) far-red light (FR), (3) a short night-break of red followed by far-red light during an otherwise dark night (R FR), (4) a short night-break of red, far-red and finally red light during an otherwise dark night (R FR R), (5) dim white light (L) and (6) red light (R). The treatments L and R resulted in significantly more severe ozone-induced visible foliar injuries relative to D and FR treatments, indicating a phytochrome-mediated response. The night-breaks resulted in a photoreversible and significantly different ozone response depending on the light quality of the last light interval (R FR or R FR R), supporting a photoreversible (between P_r and P_fr) phytochrome signalling response. Thus, in T. subterraneum, the outcome of oxidative stress due to ozone appears to depend on the photoperiod mediated by the night-time conformation of phytochrome.     

The role of calcium cations in the mechanism of phytochrome-dependent regulation of the <Emphasis Type="Italic">sdh1-2</Emphasis> gene expression and succinate dehydrogenase activity in maize leaves

The regulation of succinate dehydrogenase (SDH) activity and its gene expression by the phytochrome system has been demonstrated: red light (660 nm) reduces the SDH activity and the level of expression of the sdh1-2 gene. At the same time, the content of calcium cations in the nuclear fraction increases; it seems to be one of the mechanisms of phytochrome signal transduction in plant cells. Far-red light (730 nm) had opposite effects, i.e., increased SDH activity and the level of expression of the sdh1-2 gene. The data suggest that the SDH activity can be regulated at the level of expression in the green leaves of Zea mays by the phytochrome system with the involvement of calcium ions as a signal transduction messenger.