INTRACELLULAR DICHROIC ORIENTATION OF THE BLUE LIGHT-ABSORBING PIGMENT AND THE BLUE-ABSORPTION BAND OF THE RED-ABSORBING FORM OF PHYTOCHROME RESPONSIBLE FOR PHOTOTROPISM OF THE FERN Adiantum PROTONEMATA

The intracellular localization and orientation of the receptors for the blue light-induced phototropism in the fern Adianrum protonemata, phytochrome and the blue light-absorbing pigment, were investigated by combining the techniques of cell centrifugation and of microbeam irradiation with linearly polarized light. The phototropic response was induced in the cells even after they had been centrifuged basipetally to spin down the endoplasm from the apical region. When a polarized blue microbeam was given to a flank of the apical region of the protonema, the phototropic response after compensation of phytochrome effect by far-red light was most effectively induced when the polarization plane was parallel to the long axis of the cell. If protonemata were pre-irradiated with blue and far-red light, the phototropic response was mediated through phytochrome alone. If such pre-irradiated protonemata were similarly irradiated with a polarized blue microbeam, polarized light vibrating parallel to the cell axis was again most effective in inducing the response. These results indicate that both the blue light-absorbing pigment and the phytochrome responsible for the blue light-induced phototropism in Adiantum are confined to the plasma membrane and/or the ectoplasm and that the transition moments of their blue-absorption bands are nearly parallel to the cell surface.     

Phytochrome-mediated Phototropism in Adiantum Protonemata II. Participation of Phytochrome Dark Reversion

A microbeam irradiation technique was used to analyze phytochrome-mediated phototropism of the protonema of the fern Adiantum capillus-veneris. One side of the sub-apical zone of a dark-adapted protonema was irradiated with a red-light (R) microbeam to induce phototropic curvature toward the irradiated side. Except the cases where fluence-response relationships were examined, the protonema was stimulated with the microbeam for about 30 s to provide a fluence (5.3–5.5 mmol m^-2) optimal for curvature. When the whole protonema was pretreated with a high fluence of R (about 9 mmol m^-2), no significant curvature could be induced by immediately subsequent one-sided microbeam stimulation. It was found, however, that curvature became inducible progressively as the time of microbeam stimulation was delayed after the R pretreatment. The protonema gained nearly full responsiveness within 40 min after the pretreatment. Moreover, the inductive effect of microbeam R escaped rapidly from the reversing effect of far-red light. These results indicated that most of the far-red-absorbing form of phytochrome (Pfr), which mediates the phototropic response, undergoes relatively fast dark reversion to the red-absorbing form. The dark reversion kinetics was analyzed further by taking into account that the phototropic responsiveness after the R pretreatment was measured in the presence of background Pfr over the protonema. This analysis revealed a rate constant of about 0.002 s^-1 (t_1/2? 6 min) for the dark reversion. It is considered that the Pfr dark reversion plays a role in establishing a lateral Pfr gradient in the unilaterally irradiated protonema.     

PHOTOTROPISM AND AXIS EXTENSION IN LIGHT-GROWN MUSTARD (Sinapis alba L.) SEEDLINGS‡

Abstract— The relationship between phototropism and axis extension was examined in light-grown mustard (Sinapis alba L.) seedlings using the low pressure sodium lamp (SOX)? technique to eliminate growth responses due to phytochrome. Addition of blue light caused no net inhibition of hypocotyl elongation, but plants showed a phototropic response. Curvature was caused by a simultaneous inhibition of growth on the illuminated side of the hypocotyl and an acceleration on the shaded side. Phototropism thus occurs independently of axis elongation and suggests that they are two separate processes. The results are inconsistent with the Blaauw theory of phototropism.     

ACTION SPECTRUM FOR ENHANCEMENT OF PHOTOTROPISM BY Arabidopsis thaliana SEEDLINGS

Fluence-response relationships have been measured at wavelengths from 350 to 760 nm for the enhancement of phototropism in Arabidopsis thulium L. (Heynh) strain “Estland” by an irradiation at each of these wavelengths, given 2 h prior to a 450 nm inductive unilateral irradiation. Action spectra have been constructed from these fluence-response relationships based on: (i) the fluence required to obtain a curvature of 25° (corresponding to an enhancement of 15°), (ii) the fluence required to obtain 50% of the maximum enhancement and (iii) the fluence threshold for enhancement by a pre-irradiation. The action spectra exhibit two maxima, one at 669 nm and a second at 378 nm. The height of the maximum at 669 nm is approximately 4 times the height of the maximum at 378 nm. Based on the action spectra, it is concluded that the enhancement of phototropism in A. thaliana is mediated by phytochrome.     

PHYSIOLOGICAL CHARACTERIZATION OF A HIGH-PIGMENT MUTANT OF TOMATO

Abstract— A high-pigment (hp) mutant, which shows exaggerated phytochrome responses and three other genotypes of Lycopersicon esculenrum Mill. cv. Ailsa Craig: the aurea (au) mutant deficient in the bulk light-labile phytochrome (PI) pool, the au, hp double mutant, and their isogenic wild type, were used in this study. Measurements of phytochrome destruction in red light (R) revealed that the exaggerated responses of the hp mutant are not caused by a higher absolute phytochrome level or a reduced rate of phytochrome destruction. Fluence-response relationships for anthocyanin synthesis after a blue-light pretreatment were studied to test if the hp mutant conveys hypersensitivity to the far-red light (FR)-absorbing form of phytochrome (Pfr), i.e. the threshold of Pfr required to initiate the response is lower. The response range for the hp mutant and wild type was identical, although the former exhibited a 6-fold larger response. Moreover, the kinetics of anthocyanin accumulation in continuous R were similar in the wild-type and hp-mutant seedlings, despite the latter accumulating 9-fold more anthocyanin. Since the properties of phytochrome are the same, the hp mutation appears to affect the state of responsiveness amplification, i.e. the same amount of Pfr leads to a higher response in the hp mutant. We therefore propose that the hp mutation is associated with an amplification step in the phytochrome transduction chain. Escape experiments showed that the anthocyanin synthesis after different light pretreatments terminated with a R pulse was still 50% FR reversible after 4–6 h darkness, indicating that the Pfr pool regulating this response must be relatively stable. However, fluence-rate response relationships for anthocyanin synthesis and hypocotyl growth induced by a 24-h irradiation with 451, 539, 649, 693, 704 and 729 nm light showed no or a severely reduced response in the au and au, hp mutants, suggesting the importance of PI in these responses. We therefore propose that the capacity for anthocyanin synthesis (state of responsiveness amplification) could be established by PI, while the anthocyanin synthesis is actually photoregulated via a stable Pfr pool. The Hp gene product is proposed to be an inhibitor of the state of responsiveness amplification for responses controlled by this relatively stable Pfr species.     

Phytochrome structure: A new methodological approach

Higher plants use the protein phytochrome as a photosensor. In physiological temperatures phytochrome exists in two forms: Pr and Pfr. The chromophore of phytochrome is an open-chain tetrapyrrole. On the pathway from Pr to Pfr four intermediates (Lumi-R, Meta-Ra, Meta-Rb, and Meta-Rc) can be distinguished, while only two (Lumi-F and Meta-F) can be seen on the way back from Pfr to Pr. We have used the x-ray structure of the C-Phycocyanin protein Fremyella diplosiphon bacteria as a template to build a model (∼200 atoms) that includes only the chromophore and five amino acids of the phytochrome (Arg316–Cys321–His322–Leu323–Gln324) around it. Using the existing experimental evidences, we have proposed a three-dimensional (3D) structure for Pr, Pfr, and intermediates and a mechanism for the photoisomerization as well. Structures were fully optimized using AM1 (Unichem package on a Cray J90-NACAD). Using the INDO/S method of Zerner and co-workers, we calculated the absorption spectra of the model compounds and compared them with the experimental data. The oscillator strength ratio is an indicator of the chomophore conformation in biliproteins. The calculated spectra reproduces well the spectra of the phytochrome (Pr, Pfr, and intermediates) except for the lower energy band. This result is attributed to the small number of amino acids in the models. The calculated ratios (f_VIS/f_UV−f_osc of visible band over f_osc of UV band and f₂/f₁−f_osc of second absorption band over f_osc of first absorption band) for the models match very well the experimental ratios obtained for the phytochrome (Pr, Pfr, and intermediates). This supports the proposed mechanism for the photoisomerization process. © 1998 John Wiley & Sons, Inc. Int J Quant Chem 70: 1145–1157, 1998     

PHYTOCHROME PHOTOCONVERSION in vivo. EFFECT OF THE INITIAL Pfr/Ptot RATIO

The equation for phytochrome photoconversion, derived from photoconversion kinetics of purified phytochrome, predicts that the rates of photoconversions starting from low and high Pfr/Ptot₍₀₎, (initial Pfr/Ptot) should be the same for light of the same quality and fluence rate. The situation might be different in vivo. Phytochrome photoconversion rates were measured in excised cotyledons of Cucurbita pepo L. exposed to BL (blue; ?_BL= 0.39; ?, Pfr/Ptot at photoequilibrium) and RI (mixture of red and far red; ?_RI= 0.46) after saturating preirradiations with red and far-red to establish high (0.78) and low (0.02) Pfr/Ptot₍₀₎, respectively. Under BL, the rate of photoconversion is faster when starting from a high than a low Pfr/Ptot₍₀₎; under RI, the rate of photoconversion is faster when starting from a low than a high Pfr/Ptot₍₀₎., No effects of Pfr/Ptot₍₀₎, on photoconversion rates were found in phytochrome solutions exposed to BL and RI. These data provide another indication of the discrepancies between phytochrome photconversion kinetics in vivo and in vitro.     

AN ACTION SPECTRUM IN THE BLUE and ULTRAVIOLET FOR PHOTOTROPISM IN ALFALFA*

Abstract Phototropism is a common property of plants, but it is not known if different species use the same photoreceptor for their response. We have determined fluence-response relations for phototropism in response to brief, broad-band blue irradiation for four plant species grown under red light (Amaranthus paniculatus, Linum usitatissimum, Vigna radiata and Medicago sativa) and compared these to ones previously obtained for Pisum sativum and Zea mays, grown under similar conditions. Curves for all species showed a bell-shaped dependence on fluence, a characteristic of first positive curvature as originally defined for the Avena coleoptile, and had a similar optimal fluence, near 3 H.mol m^?2. We have obtained an action spectrum in the blue and UV spectral regions for first positive phototropism of the hypocotyl of alfalfa grown under red light. Fluence-response curves at wavelengths between 300 and 500 nm were nearly identical in shape and magnitude; whereas below 300 nm, their slopes and maximum curvatures were reduced. The action spectrum showed that activity rose sharply at wavelengths below 500 nm, peaked at 450 nm with shoulders on either side of that peak, and had lesser peaks at 380 and, in the far ultraviolet, at 280 nm. This action spectrum was very similar to ones in the literature (obtained between 350 and 500 nm) for first and second positive phototropism of oat coleoptiles. We conclude that the same photoreceptor mediates phototropism in oat and alfalfa.     

DIRECT MEASUREMENT OF PHYTOCHROME PHOTOCONVERSION INTERMEDIATES AT HIGH PHOTON FLUENCE RATES

A custom-built modulated split-beam spectrophotometer has been used to measure the absorbance of tissue samples and purified phytochrome whilst exposing the sample to actinic 633 nm laser radiation at fluence rates approaching those of daylight. This approach has allowed the direct observation of the accumulation of phytochrome photoconversion intermediates at high fluence rates. At ca 1250 μmol m^?2 s^?1 upwards of 35% of the total phytochrome was present in the form of photoconversion intermediates in tissues of maize, sunflower and tomato. In other tissues tested (wheat, bean and Amaranthus) and in purified oat phytochrome, rather smaller levels of intermediates accumulated. Upon “lights-off” only a proportion of the accumulated intermediates decayed to far-red absorbing phytochrome (Pfr), the remainder appearing as the red-absorbing form (Pr). Difference spectra suggested that, at high light levels, Pr may be reformed via a photochemical back-conversion of an intermediate in the Pr—Pfr pathway, although the involvement of intermediates in the Pfr—Pr pathway cannot be excluded. The implications of the results for the ecological function of phytochrome are discussed.     

PHYTOCHROME-MEDIATED PHOTOTROPISM IN PROTONEMATA OF THE MOSS Ceratodon purpureus BRID

Abstract— Protonemata of the moss Ceratodon purpureus cultured in white light were transferred to darkness for 3 days and then used for phototropic experiments. Irradiation of the apical region of vertically position protonemata with small beams (0.2 mm) of red light induced a growth response towards the irradiated side (positive phototropism). The phototropic response showed irradiance dependence. The effect of red light was completely reversed by far-red light following red light irradiations, demonstrating that phytochrome was the photoreceptor pigment. Far-red light or UV-blue light had no influence on either bulging or phototropism. Experiments with linearly polarized red or far-red light showed a different dichroic distribution of phytochrome in its different forms, the red-absorbing form, P_r and the far-red-absorbing form, P_fr. Red light with a vibration plane parallel to the long axis of the filaments was most effective. The effectiveness of far-red light was expressed best when its vibration plane was 90° to the electrical vector of the inductive red light.     

STUDIES ON PHOTOTROPISM OF YOUNG SPORANGIOPHORES OF PILOBOLUS KLEINII*

Abstract— Young sporangiophores of the fungus, Pilobolus kleinii, respond to unilateral illumination by bending or by growing toward light of wavelengths between 312 and 530 mμ, with peaks of sensitivity near 360 and 450 mμ. Young sporangiophores exhibit a negative phototropic response to wavelengths shorter than 300 mμ, with a strong negative response at 280 mμ. Since the action spectrum did not correspond to the absorption spectrum of the pigmented zone as measured in vivo, and since colorless sporangiophores formed on media containing diphenylamine were capable of phototropic response, it is unlikely that the conspicuous orange-yellow pigment in young sporangiophores is the photoreceptor for phototropism. The results of probing with small beams of light and the behavior of sporangiophores submerged in mineral oil, together with measurements of the refractive index of the tip and base indicate that the photosensitive region is located in the tip of the young sporangiophore.     

LIGHT PROMOTION OF SEED GERMINATION IN Datura ferox IS MEDIATED BY A HIGHLY STABLE POOL OF PHYTOCHROME

Abstract— The duration of the far-red light-absorbing form of phytochrome (Pfr) of the photoreceptor pool involved in the control of seed germination was investigated for Datura ferox seeds. These seeds require both Pfr and alternating temperatures (20/30°C) to germinate. After 24 h imbibition (25°C), the seeds received pretreatment-light pulses providing different phytochrome photoequilibria (Pfr/P), followed by a 24 h dark incubation (25°C), and test-light pulses providing different Pfr/P immediately prior to transfer to alternating temperatures. Germination increased with increasing Pfr/P provided by the test-light pulses, but was unaffected by the pretreatment-light pulses. This suggests that phytochrome synthesis, phytochrome degradation and phytochrome-mediated changes in response to phytochrome were negligible. In other experiments, red light-pretreatment pulses were followed by dark incubations (25°C) of different duration before transfer to alternating temperatures. The proportion of Pfr remaining after the 25°C incubation period was estimated by comparing germination rates with those of seeds that received test-light pulses of known calculated Pfr/P immediately prior to the start of the cycles of alternating temperatures. More than 80% of the Pfr established by a Pfr/P= 0.87 light pulse was present and active even after 48 h dark incubation at 25°C. Surprisingly, when a pretreatmentlight pulse providing a Pfr/P= 0.70 was given, the reduction in [Pfr] was significantly faster.
Germination of Datura ferox seeds is under the control of a highly stable (type II like) phytochrome pool. Apparently, this pool follows Pfr dark reversion to the red light-absorbing form, the times to reach half the original Pfr pool being > 96 h or <14 h after light pulses providing Pfr/P= 0.87 or 0.70, respectively.     

A Spectroscopy-based Methodology for Rapid Screening and Characterization of Phytochrome Photochemistry in Search of Pfr-favored Variants

Phytochromes are photosensitive proteins with a covalently bound open-chain chromophore that can switch between two principal states: red light absorbing Pr and far-red light absorbing Pfr. Our group has previously shown that the bacteriophytochrome from Xanthomonas campestris pv. campestris (XccBphP) is a bathy-like phytochrome that uses biliverdin IXα as a co-factor and is involved in bacterial virulence. To date, the XccBphP crystal structure could only be solved in the Pr state, while the structure of its Pfr state remains elusive. The aims of this work were to develop an efficient screening methodology for the rapid characterization and to identify XccBphP variants that favor the Pfr form. The screening approach developed here consists in analyzing the UV-Vis absorption behavior of clarified crude extracts containing recombinant phytochromes. This strategy has allowed us to quickly explore over a hundred XccBphP variants, characterize multiple variants and identify Pfr-favored candidates. The high-quality data obtained enabled not only a qualitative, but also a quantitative characterization of their photochemistry. This method could be easily adapted to other phytochromes or other photoreceptor families.     

PHYTOCHROME-MEDIATED PHOTOTROPISM AND DIFFERENT DICHROIC ORIENTATION OF Pr AND Pfr IN PROTONEMATA OF THE FERN ADIANTUM CAPILLUS-VENERIS L.

Abstract— Single-celled protonemata of Adiantum capillus-veneris were cultured under continuous red light for 6 days and then in the dark for 15 h. Brief local exposure of a flank (5 times 20 /mi) of the subapical region of a protonema to a microbeam of red light effectively induced a phototropic response toward the irradiated side. The degree of the response was dependent upon the fluence of the red light. Red/far-red reversibility was typically observed in this photoreaction, showing that phytochrome was the photo-receptive pigment. When the flank was irradiated with a microbeam of linearly polarized red and far-red light, red light with an electrical vector parallel to the cell surface was most effective. However, the far-red light effect was most prominent when its electrical vector was normal to the cell surface. These polarized light effects indicate the different dichroic orientation of P_r (red-light-absorbing form of phytochrome) and P_r (far-red-light-absorbing form of phytochrome) at the cell flank.     

ANALYSIS OF PHOTOTRANSFORMATION INTERMEDIATES IN THE PATHWAY FROM THE RED-ABSORBING TO THE FAR-RED-ABSORBING FORM OF AVENA PHYTOCHROME BY A MULTICHANNEL TRANSIENT SPECTRUM ANALYZER

Phototransformation of the red-absorbing form of phytochrome (Pr) to the far-red-absorbing form (Pfr) was followed with a custom-built transient spectrum analyzer. Large phytochrome, which consisted of approximately 120000-dalton monomers, was immunopurified or conventionally purified from etiolated oat (Avena sativa L., cv. Garry) shoots. Phototransformation was initiated by exciting Pr with a 115-mJ, 600-ns half-width, 655-nm laser pulse. Absorption spectra were recorded on a microsecond time scale at predetermined times after the flash. It has been reported earlier that flash excitation of large oat Pr produces a transformation intermediate with maximum absorbance near 700 nm in a difference spectrum and that this intermediate decays by two kinetically distinct reactions. Difference spectra for these two reactions are indistinguishable. Both show bleaching centered at 690 nm with no detectable associated absorbance increase between 570 and 830 nm. Subsequent appearance of absorbance at 724 nm, which presumably but not necessarily represents the appearance of Pfr, had earlier been shown to occur by two kinetically distinct reactions for large oat phytochrome. Data presented here indicate in addition the occurrence of a third, slower reaction. Difference spectra for the two faster reactions are indistinguishable, both with maxima near 728 nm and minima near 650 nm. The difference spectrum for the slowest component, however, was qualitatively different exhibiting a maximum near 722 nm with no corresponding minimum. About 15-20% of the absorbance increase at 724 nm occurred by this slowest reaction, which exhibited a half-life of 3 s at 25°C and a Q₁₀ of 1.2 for immunopurified and 1.5 for conventionally purified phytochrome. The percentage occurring by this reaction was independent of temperature over the range studied (1-25dEC). For immunopurified phytochrome the enthalpy of activation, Gibbs free energy of activation, and entropy of activation of this slowest reaction were found to be about lOkJ-mol^-1, 75kJ.mol^-1, and -220 J.mol^-1 K^-1, respectively, and for conventionally purified phytochrome 25kJ.mol^-1, 75kJ.mol^-1and —170 J.mol^-1 K^-1, respectively. The thermodynamic characteristics of this reaction indicate that it may involve a significant ordering of the protein moiety as it transforms to Pfr.     

SPECTRAL CHARACTERIZATION OF HIGH-MOLECULAR-WEIGHT PHYTOCHROME*

Abstract— Previous information about the spectral and photochemical properties of phytochrome in vitro has apparently been determined in large part with chromopeptides derived from the native molecule by proteolysis. Characterization of high-mol-wt phytochrome in vitro has led to the observation that the far-red-absorbing form (Pfr) may undergo relatively large and reversible changes in far-red extinction. Phytochrome preparations which exhibit reduced far-red extinction as Pfr also exhibit a rapid reappearance of red absorbance after discontinuing the red illumination used to establish photostationary equilibrium. This rapid change in the red spectral region is not accompanied by any equivalent absorbance change in the far-red. The molecular basis for these newly reported spectral properties is not known. However, both properties may be eliminated by the addition of either 2-mercaptoethanol or ethylenediaminetetraacetic acid.     

PHYTOCHROME and PROTEIN PHOSPHORYLATION

The molecular mode of signal transduction triggered by phytochrome is unknown. One characteristic structural/topographic feature of the physiologically active form (Pfr) of phytochrome is that its tetrapyrrole chromophore becomes preferentially exposed in the Pfr form (compared to the Pr form). Phytochrome in its Pfr form appears to affect phosphorylation of cellular proteins. The literature on the phytochrome-mediated protein phosphorylation has been reviewed in an attempt to search for the role of the chromophore topography of phytochrome in the signal transduction process. In order to initiate a dephosphorylation-phosphorylation cascade as a possible step for the signal transduction, it may interact with a cellular protein kinase to inhibit its activity. This hypothesis has been reviewed with results from phosphorylation inhibition assays by the Pfr form of phytochrome and in light of the inhibition of protein kinase activity by tetrapyrroles in general.     

KINETIC ANALYSIS OF A VERY RAPIDLY REVERTING POPULATION OF HIGH-MOLECULAR-WEIGHT PHYTOCHROME*

Abstract— Purified high-mol-wt phytochrome preparations in the absence of ethylenediaminetetraacetic acid or 2-mercaptoethanol may exhibit lower far-red extinction in the far-red-absorbing form (Pfr) than has previously been reported. Kinetic analyses suggest that such preparations of phytochrome in the Pfr form consist either of two populations of Pfr, one with normal extinction and one with greatly reduced far-red extinction, or of one population of Pfr in which each molecule possesses both one or more high far-red extinction chromophores and one or more low far-red extinction chromophores. The low-extinction form of Pfr undergoes reversion to the red-absorbing form of phytochrome with a half-life of approximately 2 min at 3°C and may represent as much as 35 per cent of the total Pfr present.     

Solid‐State NMR Spectroscopy to Probe Photoactivation in Canonical Phytochromes

The photoreceptor phytochrome switches photochromically between two thermally stable states called Pr and Pfr. Here, we summarize recent solid‐state magic‐angle spinning (MAS) NMR work on this conversion process and interpret the functional mechanism in terms of a nano‐machine. The process is initiated by a double‐bond photoisomerization of the open‐chain tetrapyrrole chromophore at the methine bridge connecting pyrrole rings C and D. The Pr‐state chromophore and its surrounding pocket in canonical cyanobacterial and plant phytochromes has significantly less order, tends to form isoforms and is soft. Conversely, Pfr shows significantly harder chromophore–protein interactions, a well‐defined protonic and charge distribution with a clear classical counterion for the positively charged tetrapyrrole system. The soft‐to‐hard/disorder‐to‐order transition involves the chromophore and its protein surroundings within a sphere of at least 5.5 Å. The relevance of this collective event for signaling is discussed. Measurement of the intermediates during the Pfr → Pr back‐reaction provides insight into the well‐adjusted mechanics of a two‐step transformation. As both Pr → Pfr and Pfr → Pr reaction pathways are different in ground and excited states, a photochemically controlled hyper‐landscape is proposed allowing for ratchet‐type reaction dynamics regulating signaling activity.