Heterologous expression and characterization of recombinant phytochrome from the green alga Mougeotia scalaris

The full-length apoprotein (124 kDa) and the chromophore-binding N-terminal half (66 kDa) of the phytochrome of the unicellular green alga Mougeotia scalaris have been heterologously expressed in the methylotrophic yeast Pichia pastoris. Assembly with the tetrapyrrole phycocyanobilin (PCB) yielded absorption maxima (for the full-length protein) at 646 and 720 nm for red- and far-red absorbing forms of phytochrome (Pr and Pfr), respectively, whereas the maxima of the N-terminal 66 kDa domain are slightly blueshifted (639 and 714 nm, Pr and Pfr, respectively). Comparison with an action spectrum reported earlier gives evidence that in Mougeotia, as formerly reported for the green alga Mesotaenium caldariorum, PCB constitutes the genuine chromophore. The full-length protein, when converted into its Pfr form and kept in the dark, reverted rapidly into the Pr form (lifetimes of 1 and 24 min, ambient temperature), whereas the truncated chromopeptide (66 kDa construct) was more stable and converted into Pr with time constants of 18 and 250 min. Also, time-resolved analysis of the light-induced Pfr formation revealed clear differences between both recombinant chromoproteins in the various steps involved. The full-length phytochrome showed slower kinetics in the long milliseconds-to-seconds time domain (with dominant Pfr formation processes of ca 130 and 800 ms), whereas for the truncated phytochrome the major component of Pfr formation had a lifetime of 32 ms.     

ON THE SIGNAL-TRANSDUCTION CHAINS OF TWO Pfr- MEDIATED SHORT-TERM PROCESSES: INCREASE OF ANCHORAGE and MOVEMENT OF Mougeotia CHLOROPLASTS

Abstract— We examined two published hypotheses on the signal-transduction chain of the light-oriented chloroplast movements in the fresh-water alga Mougeotia. One hypothesis postulates a Ca²+-influx controlled by a tetrapolar gradient of phytochrome in its far-red light absorbing form (Pfr). The other hypothesis postulates anchorage sites for actin-filaments even at those areas of the plasmalemma where phytochrome is in its inactive form (Pr). Calmodulin and Ca²+-sequestering vesicles are assumed to be essential links of this transduction chain.
To test these hypotheses we have studied the effects of Ca²+-entry blockers, Ca²+ deficiency and calmodulin antagonists on chloroplast movements and on chloroplast anchorage. None of our results support the Ca²+/calmodulin hypotheses mentioned above. The results and their implications (with regard to the role of Ca²', calmodulin and anchorage sites) are discussed.     

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.     

ULTRAVIOLET RESONANCE RAMAN SPECTRA OF PHYTOCHROME: A COMPARISON OF THE ENVIRONMENTS OF TRYPTOPHAN SIDE CHAINS BETWEEN RED LIGHT-ABSORBING AND FAR-RED LIGHT-ABSORBING FORMS

Ultraviolet resonance Raman spectra of phytochrome in the red light-absorbing form (Pr) and the far-red light-absorbing form (Pfr) are reported. The spectra excited at 240-nm provide structural information about the protein part of phytochrome. The protein contains only a very small amount of β-sheet structure and most of the tyrosine side chains are located in hydrophobic environments. Indole rings of tryptophan (Trp) interact with neighboring groups in the Pr form and these interactions become weaker with the conversion from Pr to Pfr. Some Trp side chains of Pfr are surrounded by aliphatic groups but such is not the case in Pr. These changes in the environment occur at the same time as changes in orientation of Trp side chains. Our observations suggest that interactions between Trp residues and the tetrapyrrolic chromophore occur in the Pr form and that the strength of these interactions diminishes in the Pfr form.     

PERMEABILITY AND STRUCTURAL CHANGES INDUCED BY PHYTOCHROME IN LIPID VESICLES

Abstract— This paper describes a method for rapidly monitoring early changes in electrolyte permeability induced by phytochrome from salt-loaded liposomes. The method allows for the continuous monitoring of low-level ion efflux from liposomes by measuring the conductivity of a liposome suspension medium which has osmotic and chemical potentials that promote a slow. passive efflux of the compartmented electrolytes. The addition of the far-red absorbing form of phytochrome (Pfr) to this system at 20°C immediately produces efflux rates which are 2–3 times greater than if the red-absorbing form (Pr) is added. This differential effect is not evident at 4°C and varies with the lipid composition of the liposomes. Under conditions in which Pfr induces a 2-fold greater change in the electrolyte permeability of liposomes than Pr. only about 18% more ¹²⁵I-labeled Pfr than ¹²⁵I-labeled Pr binds to the liposomes. At equimolar concentrations. the photochromic small peptide of phytochrome (60 000 dalton monomer) and the more native'large'phytochrome (120000 dalton subunits) induced equivalent changes in the electrolyte permeability of liposomes. No differential leakage of ATP, glucose, or trvpsin from liposomes was observed after Pr and Pfr reacted with vesicles enclosing these substances. The Pfr form of phytochrome promoted greater turbidity in liposome suspensions and a greater degree of aggregation and/or vesiclc fusion than Pr. The kinetics of these changes suggested that they were not the hasis of the differential permeability effects of Pr and Pfr.     

PHOTOTRANSFORMATIONS OF PHYTOCHROME

Abstract— –Phytochrome is the photoreversible chromoprotein that controls many aspects of plant growth and development Phototransformations of the red absorbing form (Pr) and the far red absorbing form (Pfr) involve initial photoreactions followed by dark relaxation reactions. Techniques for the study of intermediates of phototransformation and the present picture of intermediates involved in the phototransformations of Pr and Pfr are outlined. The molecular natures of the phototransformations are reviewed in relationship to knowledge of the chemistry of the chromophore and apoprotein. The significance of phytochrome intermediates in understanding the physiology of phytochrome controlled responses is discussed.     

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.     

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.     

RED-BLUE-INTERACTION IN Mesotaenium CHLOROPLAST MOVEMENT: BLUE SEEMS TO STABILIZE THE TRANSIENT MEMORY OF THE PHYTOCHROME SIGNAL

Abstract— Fast chloroplast orientation in Mesotaenium from profile position to face position cannot be induced by either red (R) or blue (BL) light (in contrast to Mougeotia ). Rather interaction of light signals mediated by phytochrome and blue-light photoreceptor(s) is essential for the response. If both light treatments are separated in time, the irradiation sequence R-BL is much more effective than BL-R, i. e. a gradient of the far-red (FR)-absorbing form of phytochrome (Pfr) renders following BL highly effective, but BL cannot increase the responsiveness to following Pfr. The memory of a R irradiation before BL holds only for some minutes, indicating that the physiological activity of Mesotaenium -Pfr and its photoproducts is very short-lived. This transient signal mediated by Pfr can be transformed to a more stable internal signal by interaction with BL. The interaction process does not occur at the level of photoperception. Rather, early products of the phytochrome-initiated signal transduction chain interact with excited cryptochrome or an early product of it; Pfr can be removed by FR before the onset of BL. The internal signal stores the directional information of the Pfr-gradient, so that BL is now fully effective and induces chloroplast movement.     

Spectroscopic detection of a phytochrome-like photoreceptor in the myxomycete Physarum polycephalum and the kinetic mechanism for the photocontrol of sporulation by Pfr.

Sporulation of the true slime mold Physarum polycephalum (Myxomycetales) can be triggered by the far-red/red reversible Physarum phytochrome. Physarum plasmodia were analyzed with a purpose-built dual-wavelength photometer that is designed for phytochrome measurements. A photoreversible absorbance change at 670 nm was monitored after actinic red (R) and far-red (FR) irradiation of starved plasmodia, confirming the occurrence of a phytochrome-like photoreceptor in Physarum spectroscopically. These signals were not found in growing plasmodia, suggesting the Physarum phytochrome to be synthesized during starvation, which makes the cells competent for the photoinduction of sporulation. The photoconversion rates by R and FR light were similar in the phytochromes of Physarum and etiolated oat shoots. In dark-grown Physarum plasmodia that had not been preexposed to any light only R induced a detectable absorbance change while FR did not. This indicates that most (at least 90%) of the photoreversible pigment occurs in the red-absorbing form. Since the effectiveness of FR in triggering sporulation was enhanced by preirradiation with R, it is concluded that at least part of the Pr can be photoconverted to the active Pfr photoreceptor species. We propose a kinetic mechanism for the photocontrol of sporulation by photoconversion of Pfr, which may also hold for the high-irradiance response to FR in Arabidopsis and Cuscuta.     

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.     

THE USE OF A COMPUTERIZED SPECTRORADIOMETER TO PREDICT PHYTOCHROME PHOTOEQUILIBRIA UNDER POLYCHROMATIC IRRADIATION

Abstract A method is described for predicting the effect of polychromatic irradiation upon the photo-stationary equilibrium of the plant photoreceptor phytochrome. This method follows from the rate equations for phototransformation and utilizes the in vivo action spectra for phytochrome phototransformation (Pratt and Briggs, 1966). A scanning spectroradiometer interfaced with a microcomputer is used to determine a spectral photon distribution from 360 to 800 nm. The products of the photon fluence rate and the relative quantum efficiencies at 2-nm intervals are summed over the entire visible range to yield a predicted percentage of the pigment in the Pfr form. This value was determined under eight different polychromatic light sources and was generally within 7% Pfr of the value measured in etiolated corn coleoptiles under the same light sources.     

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.     

INTERPRETATION OF THE 'DICHROIC ORIENTATION' OF PHYTOCHROME

The dichroic orientation of phytochrome observed both in the phytochrome-mediated phototropism in Adiantum protonemata and in the phytochrome-mediated chloroplast movement in Mougeotia were analyzed in terms of the orientation of the transition moment associated with the long-wavelength absorption band, assuming that phytochrome, associated with the plasma membrane, rotates around the normal to the membrane. The orientation of the long-wavelength transition moment of the phytochrome chromophore was calculated using the zero-differential overlap approximation of the molecular orbital theory for ir-electrons. The results indicate that the orientation of the long-wavelength transition moment mainly changes later than 2 ms after red light excitation of P_r, and that the different dichroic orientations of P_r and P_fr can be attributed to the change in the angle of the long-wavelength transition moment of phytochrome with the plasma membrane from 18^o to 72^o during phototransformation.     

CHROMOPHORE ROTATION IN 124-kD ALTON A vena sativa PHYTOCHROME AS MEASURED BY LIGHT-INDUCED CHANGES IN LINEAR DICHROISM

Abstract— From light-induced changes in linear dichroism, we have calculated the rotation of the long-wavelength-absorbing transition moment that occurs during phototransformation of 124-kilodalton Avena sativa phytochrome. Phytochrome was purified to homogeneity and immobilized onto Sepharose beads covalently coated with antibodies against A. sativa phytochrome. Changes in linear dichroism were induced by plane-polarized red or far-red light and measured by the absorbance differences at 660 and 730 nm using a dual-wavelength spectrophotometer equipped with polarizing filters in the measuring beams. From such measurements, we calculate a rotation angle of 31^o (or 149^o) during photoconversion of Pr to Pfr and 30^o (or 150^o) during photoconversion of Pfr to Pr. These values are similar to the value of 32^o (or 148^o) reported earlier for the rotation of the transition moment of "large" A. sativa phytochrome (∽ 120 kilodalton) isolated under conditions that did not preclude post homogenization proteolysis of the 124 kilodalton molecule.     

SOME PROPERTIES OF PHOTOTRANSFORMATION OF RYE PHYTOCHROME IN VITRO

Abstract— Much of the experimental data in the phytochrome literature has been obtained using a small-molecular-weight protein fragment. Hence, several properties of phototransformation were re-examined using large-molecular-weight rye phytochrome. The kinetics of phototransformation are first-order, both for the conversion of Pr to Pfr and for the reverse reaction. The quantum yield of phototransformation was found to be 0·28 mol Einstein^-1 for the conversion of Pr to Pfr and 0·20 mol Einstein^-1 for the conversion of Pfr to Pr. Intermediates in phototransformation were measured by cycling the pigment with high-intensity mixed red and far–red light. The difference spectrum of these intermediates between 367 and 575 nm was found to be similar to that previously reported for oat and pea phytochrome. Analysis of intermediate decay indicated complex kinetics and not a single first-order species. Transient absorbancy changes in the blue region of the spectrum upon actinic illumination could be attributed to differential rates of initial bleaching of the two forms of the pigment and a consequent alteration in the proportion of the two forms in the mixture until photostationary equilibrium is re-established.     

Spectral properties of phytochrome Agp2 from Agrobacterium tumefaciens are specifically modified by a compound of the cell extract

Phytochromes are widely distributed photoreceptors that are converted by light between the red absorbing Pr and the far-red absorbing Pfr form. The soil bacterium Agrobacterium tumefaciens contains two phytochromes, Agp1 and Agp2, which act as light-regulated histidine kinases. Whereas most phytochromes are stable in the Pr form, Agp2 and few other phytochromes convert into Pfr in darkness. We have shown in a previous publication that the spectral properties of recombinant Agp2 are modified by compounds of the cell extract from an Agrobacterium agp1(-)/agp2(-) double knockout mutant. In the present work we performed concentration series which show that the interaction is specific and that the modifying factor has a concentration of ca. 0.2 microM. We have also performed a series of mixing experiments with the truncated protein Agp2-M2, which consists of the N-terminal chromophore module (501 amino acids). The cell extract inhibited the photoconversion of Agp2-M2 in an unspecific way. In concentration series, this negative effect was less pronounced when lower concentrations of Agp2-M2 were used. In the presence of excess Agp2-M2 apoprotein, the cell extract did no longer modify the spectral properties of Agp2. The data suggest that the factor of the cell extract interacts specifically with the N-terminal moiety of Agp2.     

LIGHT INDUCED FLUORESCENCE SPECTRAL CHANGES IN NATIVE PHYTOCHROME FROM Secale cereale L. AT LIQUID NITROGEN TEMPERATURE

Abstract— Fluorescence spectra of native rye phytochrome were determined under different light conditions at liquid nitrogen temperature. Fluorescence spectrum of the red-light-absorbing form (Pr) had a major peak at about 685 nm (14 600 cm⁻¹) and a broad sub-peak at about 515 nm (19 400 cm⁻¹). The peak height at 685 nm was reduced by irradiation with monochromatic light of 640 nm, and a new peak became obvious at about 702 nm (14250 cm⁻¹). This spectral change was almost completely reversed by subsequent irradiation with 700-nm light. Fluorescence spectrum of the photoequilibrium mixture of Pr and far-red-light absorbing form under continuous red light showed a sharp peak at about 685 nm having a peak height ca. 12% of Pr, and a broad sub-peak at about 508 nm (19 700 cm⁻¹). Light of 730 nm did not reduce the peak height at about 685 nm but induced a new shoulder at about 699 nm (14300 cm⁻¹). Monochromatic light of 640 and 700 nm given following the light of 730 nm could not reverse the spectral change at 699 nm induced by the irradiation with 730-nm light. Fluorescence spectrum of Pr in partially degraded phytochrome was similar to that in native phytochrome but the peak position in the red region was shifted by about 5 nm (100 cm⁻¹) to the blue.     

PHYTOCHROME ACTION: A REAPPRAISAL

Stems of fully green plants show at least two types of response to light. In one, P_fr inhibits elongation. The second is a promotion of elongation which operates only in light; the effectiveness of red and far-red wavelengths indicates that this response is also mediated through phytochrome. Studies of the de-etiolation process also provide evidence for two modes of action of phytochrome; one is a P_fr-dependent reaction, and the second requires continuous light (or frequent short irradiations). It is proposed that, in addition to reactions which require P_fr and proceed in darkness, an important reaction of phytochrome in green plants occurs only in light. We have termed these two modes of action of phytochrome “static” and “dynamic”. The static mode operates after a brief exposure to light which establishes P_fr; the potential responses are largely reversible by far-red and exhibit reciprocity. The dynamic mode operates only in light and the responses do not show reciprocity. We consider that this mode operates through the transition from one bound form of phytochrome to another. The possible involvement of these two modes of action of phytochrome in photoperiodic mechanisms is discussed.     

Phytochrome-dependent photomovement responses mediated by phototropin family proteins in cryptogam plants

In this review, we describe the regulation of photomovement responses by phototropin and phytochrome photoreceptors. The blue light receptor phototropin mediates various photomovement responses such as phototropism, chloroplast movement and stomatal opening. In cryptogamic plants including ferns, mosses and green alga, red as well as blue light mediates phototropism and chloroplast movement. The red/far-red light reversibility suggests the involvement of phytochrome in these responses. Thereby, plant growth is presumably promoted by coordinating these photomovements to capture efficiently light for photosynthesis.