A PHYTOCHROME STUDY USING TWO-LASER/TWO-COLOR FLASH PHOTOLYSIS: I700 IS A MANDATORY INTERMEDIATE IN THE PrPfr PHOTOTRANSFORMATION

Abstract— The phototransformation of native (124 kDa)oat phytochrome, P_r P_fr, Has been studied at 10C by two laser/ two-color flash photolysis. the overall P_rP_fr reaction yield did not vary with temperature within the range4–21C. Foloeing the excitation of P_r with a single 15 ns laser flash at 650nm, the formation of P_fr was quantitavely measured in a time-resolved experiment in the presence of a second 8 ns laser flash at 710 nm delayed from the initial flash. the second laser flash causes at 1.0 s after the initial laser flash a depletion of the uintermediate I₇₀₀ as welll as a reduction of the P_fr absorption at 730 nm. The depletion of I₇₀₀ correlates quantitavely with the reduction of P_fr formation. The absorpton spectra of I₇₀₀ and of the following intermendiate, I_bi, were calculated assuming that the amount of P_r, which is photoconverted by a single laser, equals the amount of P_fr formed.     

DEPENDENCE OF Pfr/Ptot-RATIOS ON LIGHT QUALITY and LIGHT QUANTITY

Abstract— Not only the spectral distribution of the light source determines the relative proportion of phytochrome in the P_fr(P_r) form, the P_fr/P_tot-ratio also depends strongly on the fluence rate of the irradiation. This dependence has been observed in the cotyledons of etiolated mustard seedlings for blue light of fluence rates below 20 Wm_-2. It has also been observed for white light and seems to be a characteristic of the phytochrome system resulting from the involvement of phytochrome thermal reactions as well as P_r P_fr photoconversions. The fluence rate dependence of P_fr/P_tot-ratios can be used to analyze the characteristic transformations of the phytochrome system. Phototransformations together with a fast thermal transformation (τ_½⋍ 3min) are consistent with the results obtained for blue and white light.     

BLUE LIGHT INDUCED PHOTOTRANSFORMATION OF PHYTOCHROME IN THE PRESENCE OF FLAVIN*

Abstract— Irradiation of the P_r form of phytochrome in the presence of flavin mononucleotide (FMN) which absorbs the actinic blue light yields P_fr at a rate greater than that in the absence of FMN. The actinic blue light absorbed by FMN enhances the phototransformation of P_r via the energy transfer from the former to the latter. On the other hand, the photoreversion of P_fr was inhibited by the presence of FMN when illuminated with blue light. The lack of photo-enhancement of the reversion of P_r, by blue light suggests that the P_fr chromophore (acceptor) transition dipole is virtually perpendicular to the FMN transition dipole, as the result of a chromophore reorientation in the P_r→P_fr phototransformation. The fact that blue light absorbed by flavin preferentially enhances the forward phototransformation of phytochrome while inhibiting the reversion may have an important implication in the high irradiance responses in plants in terms of a preferential accumulation of P_fr by blue light excitation.     

RESONANCE RAMAN SPECTRA OF THE Pr-FORM OF PHYTOCHROME

Abstract— Resonance Raman spectra of the P_r-form of oat phytochrome have been obtained at 77 K. Interference from phytochrome fluorescence is avoided by employing far-red 752 nm excitation. Vibrational assignments are suggested for the tetrapyrrole chromophore in phytochrome by comparison with previously published model compound spectra and by examining the characteristic shifts induced by deuteration of the pyrrole nitrogens. The lack of carbonyl intensity, the frequencies of the 1626 and 1644 cm^-1 C=C stretching modes, and the presence of an intense mode at 1326 cm^-1 are all consistent with a protonated structure for the tetrapyrrole chromophore in P_r. This suggests that the -50 nm red-shift of the protein-bound chromophore absorption compared to the chromophore in vitro is caused by protonation of the pyrrole nitrogen.     

CHARACTERIZATION OF A MICROSECOND INTERMEDIATE IN THE LASER FLASH PHOTOLYSIS OF SMALL PHYTOCHROME FROM OAT

Abstract— Irradiation of small phytochrome from oat in its P_r form with 15 ns laser pulses of different wavelengths(605–655 nm) gave rise to a difference absorption with maxima at 400 and 685 nm for the first detectable transient. Bleaching of a 660 nm band was observed, non-recuperable up to 1 ms. The transient absorption has a lifetime of 70±15 μs at 273 K. The transient is tentatively identified as lumi-R and the conformation of its chromophore is postulated to be more extended than that of P_r. A deviation from the exponential decay of the lumi-R absorption at 284 and 300 K and the lack of observable enhancement of the far-red absorption within 1 ms are interpreted in terms of the appearance of still other intermediates on this time scale between lumi-R and P_fr phytochrome.     

MODELS FOR THE CHROMOPHORE STRUCTURE OF THE PHOTOTRANSFORMATION INTERMEDIATES OF PEA PHYTOCHROME IN VITRO

Abstract— The changes in the chromophore structure of pea phytochrome during phototransformation in vitro from the red-light-absorbing form (P_r) to the far-red-light-absorbing form (P_fr), and from P_fr to P_r, were analysed in terms of wavelength and oscillator strength of absorption, using the zero-differential overlap approximation of the molecular orbital theory for electrons. The effect of a point-charge and a point-dipole on the optical absorption of phytochromobilin intermediates were examined using the stationary perturbation theory for degenerate states. The results indicate that the cis-trans photoisomerization of the pyrrole ring D, if any, occurred within 10 μs after a laser-flash excitation of the phytochrome, and that the conformations of phytochromobilin and the protein moiety of phytochrome were not significantly changed during the period of examination of phototransformation in either direction.     

THE TRANSFORMATION OF PHYTOCHROME TO ITS PHYSIOLOGICALLY ACTIVE FORM

Abstract— The phototransformation of phytochrome ( P_r ) to its physiologically active form (P_fr) can be best described by a tautomeric proton shift in ring A without a substantial con formational change of the chromophore. The electronic aspect of this transformation is discussed.     

CIRCULAR DICHROISM OF THE PHYTOCHROME CHROMOPHORES IN NATIVE AND DENATURED STATE

Abstract— The circular dichroism spectra of oat phytochrome were recorded. Qualitatively, the same spectra were found for large (360 kilodaltons) and small (60 kilodaltons) phytochrome. Quantitative CD data were reported for small P_r and P_fr (photoequilibrium mixture with 20% P_r) in tris buffer (native state) and in acid urea (denatured state). Further, the CD spectra of a phytochromobilinpeptide in acid solution with and without urea were recorded. Differences between the data in native and denatured state are discussed.     

COMPARATIVE PHOTOCHEMICAL ANALYSIS OF HIGHLY PURIFIED 124 KILODALTON OAT and RYE PHYTOCHROMES in vitro

Abstract A direct comparison of the photochemical interconversions between red (P_r-) and far-red (P_fr-) absorbing forms of highly-purified 124 kDa oat and rye phytochromes under identical experimental conditions was performed. In two different buffer systems at 5°C, the quantum yields for the P_r to P_tr and P_fr to P_r phototransformations under constant red and far-red illumination, φ _r and φ_fr respectively, were determined to be 0.152-0.154 and 0.060-0.065 for oat preparations and 0.172-0.174 and 0.074-0.078 for rye preparations. These values as well as the wavelength dependence of the photoequilibrium produced under continuous illumination throughout the visible and near-ultraviolet spectrum were based on the absorption spectra of the two phytochrome preparations and revised molar absorption coefficients. The molar absorption coefficients were estimated by quantitative amino acid analysis and shown to be identical for the two monocot phytochromes (i.e. 132 mM ⁻¹ cm⁻¹ at the red absorption maximum for the P_r form). Because these measurements were performed under identical experimental conditions, including buffer, temperature, light fluence rate, and instrumentation, the differences observed must reflect structural features inherent to the two different monocotyledonous phytochromes.     

CONTROL BY PHYTOCHROME OF EXTENSION GROWTH and POLAROTROPISM IN CHLORONEMATA OF Funaria hygrometrica

Abstract— The photocontrol of extension growth and polarotropism has been investigated in chloronemata of the moss Funaria hygrometrica Hedw. When grown on a 12/12 h light/dark cycle, chloronemata show light-stimulated diurnal variations in elongation rate with no evidence of a circadian rhythm. Regulation of elongation by a low energy, photoreversible phytochrome mechanism was demonstrated by brief red (R) and far red (FR) irradiations given either at the end of the day (EOD) or 6 h later as a night break (NB). Night break irradiation with polarised R caused polarotropic reorientations of chloronemal growth also through a FR-reversible, low energy phytochrome mechanism. Such transformations of P_r to P_fr were accompanied by a 90° shift in the orientation of the phytochrome chromophore. Microbeam irradiation indicated that the phytochrome chromophores involved in both responses were predominantly, if not entirely, located in the tip of the apical chloronemal cell.     

PHYTOCHROME INTERMEDIATES IN VIVO– III KINETIC ANALYSIS OF INTERMEDIATE REACTIONS AT LOW TEMPERATURE

Abstract— Kinetic experiments have provided evidence for a series of light and dark reactions of phytochrome intermediates at low temperature in Pisum epicotyl tissue. A photoequilibrium exists between P_r and P₆₉₈, and between P_fr and P₆₅₀. A dark reversion of P₆₉₈P_r and P₆₅₀p_fr at –70°C has been demonstrated. When cooled to 70°C under incandescent light, most of the phytochrome in the tissue is driven into photochemically unreactive intermediates. About 2% of the phytochrome remains as weakly absorbing intermediates that form P_r and P_fr in darkness. A scheme is presented for phytochrome phototransformation in vivo.     

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.     

Purification and Characterization of Recombinant Affinity Peptide-Tagged Oat Phytochrome A

Abstract— Full-length Avena sativa (oat) phytochrome A (ASPHYA) was expressed in the yeast Saccharomyces cerevisiae and purified to apparent homogeneity. Expression of an ASPHYA cDNA that encoded the full-length photoreceptor with a 15 amino acid'strep-tag'peptide at its C-terminus produced a single polypeptide with a molecular mass of 124 kDa. This strep-tagged polypeptide (ASPHYA-ST) bound tightly to streptavidin agarose and was selectively eluted using diaminobiotin, with a chromatographic efficiency of 45%. Incubation of ASPHYA-ST with phytochromobilin (PφB) and the unnatural chromophore precursors, phycocyanobilin (PCB) and phycoerythrobilin (PEB), produced covalent adducts that were similarly affinity purified. Both PφB and PCB adducts of ASPHYA-ST were photoactive-the PφB adduct displaying spectrophotometric properties nearly indistinguishable from those of the native photoreceptor, and the PCB adduct exhibiting blue-shifted absorption maxima. Although the PEB adduct of ASPHYA-ST was photochem-ically inactive, it was intensely fluorescent with an excitation maximum at 576 nm and emission maxima at 586 nm. The superimposability of its absorption and fluorescence excitation spectra established that a single bilipro-tein species was responsible for fluorescence from the adduct produced when ASPHYA-ST was incubated with PEB. Steric exclusion HPLC also confirmed that ASPHYA-ST and its three bilin adducts were homodimers, as has been established for phytochrome A isolated from natural sources. The ability to express and purify recombinant phytochromes with biochemical properties very similar to those of the native molecule should facilitate detailed structural analysis of this important class of photoreceptors.     

PHOTOCONVERSION OF PHYTOCHROME IN VIVO STUDIED BY DOUBLE FLASH IRRADIATION IN Mougeotia AND Avena

Abstract— The kinetics of phytochrome phototransformation from the red-absorbing form (P_r) to the far-red-absorbing form (P_fr) in vivo at 22°C were studied using a double flash apparatus with 1-ms flashes. Photoconversion by simultaneous flashes of red light saturates at a low P_fr level, indicating the possible attainment of a photoequilibrium between the excitation of P_r and the photoreversion of intermediates in the course of the I-ms flashes. At saturation energy, simultaneous flashes resulted in about 50% as much P_fr as was produced by saturating irradiation with 5 s red light. Intermediates of the phototransformation pathway were analysed by separating two red or a red and a far-red flash by variable dark intervals. In both plants phototransformation intermediates with half-lives < 1 ms occur, but they are too short-lived to characterize by our method. The subsequent intermediates have half-lives of about 7 ms and 150 ms in A vena , 2 ms and 10 ms in Mougeotia. The conversion from P_r to P_fr seems to be completed 1 s after the red flash in Avena. In the alga Mougeotia , P_fr formation seems to be finished within only 50 ms after the inducing red flash. The kinetics obtained from physiological and spectrophotometric experiments with Avena mesocotyls are almost identical. These observations indicate that the physiological response corresponds directly to the amount of P_fr produced and not to phototransformation intermediates or "cycling" between P_r and P_fr.     

OPTICAL PROPERTIES OF PHYTOCHROME IN THE BLUE- SPECTRAL REGION AS MEASURED IN VIVO AND IN VITRO

In the blue spectral region, the phototransformation difference spectrum of oat phytochrome extracted as P_fr differs from that of phytochrome extracted as P_r. The difference absorbance maximum for phytochrome extracted as P_fr is at 420 nm, while that extracted as P_r is at 412 nm. The phototransformation difference spectrum measured in the blue in oat coleoptile tips without inner leaves, corresponds very well with that of phytochrome as extracted in its P_fr form. There is, however, a slight apparent attenuation of the blue difference band relative to those in the red-far-red. In coleoptile tissue containing inner leaves, the blue difference band is relatively even more highly attenuated. A similar attenuation is observed in the blue, in the protochlorophyllide to chlorophyllide phototransformation difference spectrum. In the spectrum measured with excised coleoptile without inner leaves, there is a small attenuation, while in coleptile tissue with inner leaves the attentuation is nearly 9-fold. These data suggest that the observed attenuation is probably artifactual. Neither instrumental non-linearity nor fluorescence induced by the measuring beam could explain the observed attenuation. It is suggested that the observed attenuation is probably mainly the result of wavelength dependent scatter amplification, the amplification in the blue being attenuated by the high background absorption of other pigments in this region.     

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.     

PHYTOCHROME INTERMEDIATES IN VIVO–II. CHARACTERISATION OF INTERMEDIATES BY DIFFERENCE SPECTROPHOTOMETRY

Abstract— In epicotyl tissue of Pisum , irradiation of P_r at – 196°C forms a stable product P₆₉₈, whereas P_fr forms a stable product P₆₅₀. On warming P₆₉₈, dark transformation to P_r predominates. On warming P₆₅₀ to – 70°C an intermediate P₆₉₀ is formed which bleaches on further warming to –10°C. When tissue is cooled to –196°C during actinic irradiation, difference spectra for subsequent warming to –10°C indicate that P_r, P_fr and an intermediate P₇₁₀ are formed from weakly absorbing intermediates. Complete photoconversion of P_r to P_fr is not possible at temperatures below –5°C. As the temperature is reduced, the amount of P_fr produced from P_r decreases, while P₇₁₀ increases. P₇₁₀ can be photoconverted at –20°C and above, ultimately forming P_r, but in contrast to P_fr it is not photoconvertible at –196°C.     

PHYTOCHROME INTERMEDIATES IN VIVO-I. EFFECTS OF TEMPERATURE, LIGHT INTENSITY, WAVELENGTH AND OXYGEN ON INTERMEDIATE ACCUMULATION

Abstract— Accumulation of weakly absorbing phytochrome intermediates has been demonstrated in Pisum epicotyl tissue under conditions of pigment cycling using a quasi-continuous measuring spectrophotometer. An action spectrum shows 690–700 nm to be the most efficient wavelength range in this process. Difference spectra for the decay of intermediates maintained by 690 nm light show that, if the experiment is done at 0°C, only P_fr is formed. At – 11°C, intermediates decaying to P_r can also be observed. At – 20°C, P_r is produced as well as a pigment with peak absorption at 710nm. Kinetic analysis of intermediate decay at – 11°C reveals that at least two intermediates are maintained by 690 nm light. The level of intermediate maintained by incandescent light at 0°C was 25% higher in air than in nitrogen.     

STUDIES ON PLANT BILE PIGMENTS—9. PHOTOOXIDATION OF A-DIHYDROBILINDIONE AS PHYTOCHROME MODEL IN ACID MEDIUM

Abstract— The photooxidation of octaethyl-2, 3-dihydrobilindione ( 2 ) as a model for the P_r-chromophore has been studied in acidic methanol. When 2 is titrated with HCl to the cation and irradiated under N₂ with an excess of I₂, violin and rhodin type pigments are formed bearing methoxy-substituents at either C-4,5 or C-15,16, respectively. The reaction is slowed down as compared to neutral conditions, it is no longer regioselective at the C-5 methine bridge, and the formation of dimers is suppressed. From the data of this and earlier chemical model studies on the photooxidation of 2 , and the known properties of phytochrome, a reaction for the P_r→ P_fr phototransformation is suggested, in which the chromophore is attacked by the protein moiety in a sequence of oxidation and nucleophilic substitution, which may lead to a primary redox signal for the physiological response.     

EVIDENCE FOR A SEQUENTIAL PATHWAY FROM Pr TO Pfr OF THE PHOTOTRANSFORMATION OF 124-kDa OAT PHYTOCHROME

Abstract. Phototransformation kinetics of 124-kDa oat phytochrome at 298 K after a red (660-nm) laser flash excitation were recorded at different wavelengths. The kinetics of the dark relaxation processes for lumi-R to P_fr can be satisfactorily described by only 3 rate constants: k = 28000 s^-1 370 s^-1 and 20 s^-1. The first rate constant is due to the decay of lumi-R to meta -Ra. The latter two rate constants correspond to processes establishing the far-red (>700 nm) absorption band. No meta -Rb could be detected. From the wavelength dependency of the amplitudes of these two rates, parallel pathways in the formation of P_fr could be excluded. A unique sequential pathway for the dark relaxation leading to P_fr seems to be an intrinsic property of 124-kDa phytochrome, however. Assuming a sequential pathway, molar extinction coefficients for intermediates have been calculated. These values agree with molar extinction coefficients obtained from low-temperature spectra. The process with a rate constant of 370 s^-1 corresponds to absorbance changes for the formation of meta -Rc from meta -Ra and the rate constant of 20 s^-1 describes the absorbance changes due to the transformation of meta -Rc to P_fr.