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.     

THE PHOTOTRANSFORMATION PATHWAY OF DIMERIC OAT PHYTOCHROME FROM THE RED-LIGHT-ABSORBING FORM TO THE FAR-RED-LIGHT-ABSORBING FORM AT PHYSIOLOGICAL TEMPERATURE IS COMPOSED OF FOUR INTERMEDIATES

Abstract— –Phototransformation of oat type I phytochrome in vitro from the red-light-absorbing form (Pr) to the far-red-light-absorbing form (Pfr) at physiological temperature (24°C) was investigated with a multichannel transient spectrum analyser. Four sequential intermediates were detected between Pr and Pfr. Absorption spectra of these intermediates suggested that three of them corresponded with the intermediates lumi-R, meta-Ra and meta-Rc detected earlier at low temperature spectroscopy. A new intermediate named meta-Rb was found in the pathway between meta-Ra and meta-Rc. The new intermediate is not identical with meta-Rb previously detected at low temperature. The rate constant of Pfr appearance in isolated oat phytochrome dissolved in buffer containing 5% (vol/vol) glycerol was similar to that of etiolated pea epicotyl tissue.     

PHOTOACTIVITY OF TRANSIENT INTERMEDIATES IN THE PATHWAY FROM THE RED-ABSORBING TO THE FAR-RED-ABSORBING FORM OF Avena PHYTOCHROME AS OBSERVED BY A DOUBLE-FLASH TRANSIENT-SPECTRUM ANALYZER

Abstract— Phototransformation from the red-absorbing form of phytochrome (Pr) to the far-red-absorbing form occurs via at least two reaction stages. We report here on the photoactivity of the intermediates present during these two stages, as detected with 114/118-kdalton Avena phytochrome by a transient spectrum analyzer with two actinic lasers of microsecond duration. Flash activation of intermediates present during the first stage results in their photoconversion back to Pr within the time resolution provided by the analyzer, which is 10 μs. Since these intermediates are present within 60 ns of excitation of Pr (Cordonnier et al. , 1981), a single flash of ms duration as used here should yield a photostationary equilibrium between them and Pr. The proportion of Pr converted to intermediates by a single saturating flash is estimated to be about 30%. Flash activation of intermediates present during the second stage converts them to another transient intermediate stage, which decays thermally to Pr within 2 ms.     

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.     

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.     

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.     

Recombinant Phytochrome of the Moss Ceratodon purpureus: Heterologous Expression and Kinetic Analysis of Pr→ Pfr Conversion

Abstract— The phytochrome-encoding gene Cerpu;PHY;2 ( CP2 ) of the moss Ceratodon purpureus was heterologously expressed in Saccharomyces cerevisiae as a polyhistidine-tagged apoprotein and assembled with phytochromobilin (P φ B) and phycocyanobilin (PCB). Nickel-affinity chromatography yielded a protein fraction containing approximately 80% phytochrome. The holoproteins showed photoreversibility with both chromophores. Difference spectra gave maxima at 644/716 nm (red-absorbing phytochrome [P_r]) far-red-absorbing phytochrome [P_fr]) for the PCB adduct, and 659/724 nm for the PφB-adduct, the latter in close agreement with values for phytochrome extracted from Ceratodon itself, implying that PφB is the native chromophore in this moss species. Immunoblots stained with the antiphytochrome antibody APC1 showed that the recombinant phytochrome had the same molecular size as phytochrome from Ceratodon extracts. Further, the mobility of recombinant CP2 holophyto-chrome on native size-exclusion chromatography was similar to that of native oat phytochrome, implying that CP2 forms a dimer. Kinetics of absorbance changes during the P_r→ P_fr photoconversion of the PCB adduct, monitored between 620 and 740 nm in the microsecond range, revealed the rapid formation of a red-shifted intermediate (I₇₀o)> decaying with a time constant of - 110 u.s. This is similar to the behavior of phytochromes from higher plants when assembled with the same chromophore. When following the formation of the P_fr state, two major processes were identified (with time constants of 3 and 18 ms) that are followed by slow reactions in the range of 166 ms and 8 s, respectively, albeit with very small amplitudes.     

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.     

PHYTOCHROME STATES IN ETIOLATED PEA SEEDLINGS: FLUORESCENCE AND PRIMARY PHOTOREACTIONS AT LOW TEMPERATURES

Emission spectra of the red phytochrome form (Pr) and fluence time-response curves of the Pr fluorescence intensity changes were measured in etiolated pea seedlings at low temperatures (80–150 K) in connection with its phototransformations into the initial photoproduct (Lr) and back upon actinic red (667 nm) and far-red (696 nm) illumination. The variable fluorescence reaches 45% at 85 K and decreases with the rise of temperature. Three kinetic components of the changes were found in the direct (Pr→Lr) and back (Lr→Pr) photoreactions belonging to three states of phytochrome: “slow”, “fast” and “very fast” (respective indices: s, f and vf). The amplitudes of the components and rate constants to reach photoequilibrium were determined in the direct and back photoreactions at different temperatures, and from this, their quantum yields, extent of the Pr?Lr phototransformation and activation energy of the reactions were evaluated for the three Pr and Lr states. The yields differ from each other by approximately a factor of 10 and those for the direct and back photoreactions are close to each other. The proportion of the amplitudes of the variable fluorescence of the three phytochrome states changes with temperature and upon the Pr→Lr photo-transformation and the Pr states differ in the position of their emission spectra by 3–5 nm. A close similarity between the Pr and Lr properties was observed, which implies a symmetrical scheme of their photoreactions. It is suggested that the three phytochrome species may originate in different conformational states of the chromophore and they independently transform in parallel photoreactions into the respective photoproducts: Prvf?Lrvf, Prf?Lrf and Prs?Lrs.     

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.     

Large-scale Generation of Affinity-purified Recombinant Phytochrome Chromopeptide

Abstract— Two different yeast expression systems, Pichia pastoris and Hansenula polymorpha, are compared for their capability to express in functional form the 65 kDa N-ter-minal portion of oat phytochrome A (phyA, spanning amino acids 1-595). The front half of phytochrome was selected for this investigation because it exhibits a greater stability than the full-length protein, and it harbors full spectroscopic and kinetic properties of phytochrome, allowing an exact proof of the functional integrity of the recombinant material. In the comparison between the two expression systems used, special emphasis was given to optimizing the yield of the expression and to improving the quality of the expressed material with respect to the proportion of functional protein. From identical volumes of cell culture, H. polymorpha synthesized between 8- and 10-fold more functional protein than P. pastoris. Following the observation by Wu and Lagarias (Proc. Natl. Acad. Sci. USA 93, 8989-8994,1996) that P. pastoris endogenously produces the chromophore of phytochrome, phytochromobilin (PpHB) in significant amounts that leads to formation of spectrally active phytochrome during expression, the invention of an alternative high-yield expression system was strongly demanded. A Histag was attached to the C-terminus of the recombinant protein, which allows for a convenient and efficient purification and selects the full-length proteins over translationally truncated peptides. Fully reconstituted chromo-proteins showed an A660A280 ratio of 1.2, indicating the high degree of reconstitutable apoprotein obtained by this procedure. The assembly between apoprotein and the chromophore phycocyanobilin when followed time-resolved yielded a time constant (obs) of 35 s. The λ_max values of the red-(Pr) and the far red-absorbing (Pfr) forms of phytochrome (665 and 729 nm) of the recombinant 65 kDa chromopeptide, reconstituted with PcjiB are nearly identical to those of native full-length oat phytochrome. The kinetic parameters of the affinity-purified 65 kDa phytochrome chromoprotein for the Pr I700 Pfr conversion are compared to those of the recombinant 65 kDa chromoprotein, lacking the His-tag and to wild-type oat phytochrome. Referring to wild-type phytochrome allows determination of whether the recombinant material has lost spectral properties during the purification procedure. The decay of the primary intermediate (I₇₀₀) occurs with nearly the same time constant for the His-tagged chromoprotein and for the reference (110 and 90 mUs, respectively). The formation of the Pfr form was fitted with three exponentials in both the His-tagged and the reference chromoprotein with the middle component being slightly smaller and the longest component being remarkably larger for the His-tagged protein (1.5, 10 and 300 ms) than for the reference (1.4, 18 and 96 ms). This selective slowing down of the long kinetic component in the millisecond time range may be indicative of stronger interactions between protein domains involving the C-terminus that in the His-tagged form exhibits increased polarity.     

PHOTOREVERSIBLE Ca2+-DEPENDENT AGGREGATION OF PURIFIED PHYTOCHROME FROM ETIOLATED PEA AND RYE SEEDLINGS

The aggregation of phytochrome purified from etiolated pea ( Pisum satirum cv. Alaska) and rye ( Secale cereale cv. Cougar) tissues was investigated by centrifugation and turbidimetry. Purified pea phytochrome (A₆₆₉/A₂₈₀= 0.88), if irradiated with red light, became precipitable in the presence of CaCl₂. The precipitation upon red-light irradiation was optimal at a Ca^2- or Mg²⁺ concentration of 10–20 m M , was greater at increased phytochrome concentration or lower pH values, and was inhibited by 0.1 M KG. The precipitated phytochrome slowly became soluble after far-red light exposure.
Turbidity of pea phytochrome solutions after red-light irradiation also increased rapidly in the presence of either Ca²⁺ or Mg²⁺. Far-red light exposure after the red light cancelled the turbidity increase. Rye phytochrome showed less turbidity increase than pea phytochrome and occurred only in the presence of Ca²⁺. Partially degraded pea phytochrome produced by endogenous proteases in the extract did not show the turbidity increase. Undegraded pea phytochrome also associated with microsomal fractions under conditions similar to those described above, but the partially degraded phytochrome did not.     

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.     

STUDIES ON THE PROTEIN CONFORMATION OF PHYTOCHROME

Abstract— The extinction coefficients for large rye phytochrome were found to be: Fluorescence and circular dichroism spectra of large- and small-molecular-weight rye phytochrome give no evidence for a protein conformational change on phototransformation of phytochrome. The large molecule has a fluorescence emission peak at 331 nm when excited at 290 nm, and an excitation peak for this emission at 288 nm. The circular dichroism spectra indicate that large rye phytochrome has about 17–20% a-helix content, 30%β-structure and 50% random coil, and that the small rye phytochrome has about 10–13%α-helix content. The ultraviolet difference spectra for large and small rye phytochrome are similar and differ from the difference spectrum of the small oat phytochrome in the relative size of the 296–298 nm peak. The difference spectra may reflect changes in chromophore absorbance and in the environment of amino acid residues near the chromophore, particularly of tyrosine, and perhaps of tryptophan and cysteine.     

FLUORESCENCE QUANTUM YIELDS OF 124-kDa PHYTOCHROME FROM OAT UPON EXCITATION WITHIN DIFFERENT ABSORPTION BANDS

Abstract— A fluorescence quantum yield (emission at650–850 nm) of π= (2.3 ± 0.3)10⁻³ was measured for the red-absorbing form (Pr) of 124-kDa phytochrome from etiolated oat seedlings ( Avena sativa ) upon excitation in the Soret band at Λ^exc= 380 nm. The small difference between this value and the previously determined quantum yield with Λ^exc= 640 nm, π= (3.5 ± 0.4)10⁻³is attributed to a blue-absorbing emitter responsible for the "anomalous" or "blue" emission of the chromoprotein in the region from ca. 400 to 550 nm. The absorption of Pr at 380 nm is consequently somewhat lower than that measured directly from the spectrum. Processes from upper excited states of the Pr phytochromobilin-derived chromophore other than rapid relaxation to the emitting state are not important. A quantum yield of Φ ' 1.2 times 10⁻³ is estimated for the blue fluorescence. The proportion of the blue emitters relative to Pr appears to be relatively high.     

Determination of the chromophore structures in the photoinduced reaction cycle of phytochrome

The chromophore structures in the parent states Pr and Pfr as well as in the photocycle intermediate Lumi-R of oat phytochrome phyA are determined by comparison of the experimental resonance Raman spectra with calculated Raman spectra that have been obtained by density functional theory calculations (B3LYP) using scaled force fields. The spectra were calculated for various tetrapyrrole geometries including more than twenty different methine bridge isomers. For the parent states Pr and Pfr the best agreement in terms of vibrational frequencies, isotopic shifts, and Raman intensities was achieved with the ZZZasa and ZZEssa geometry, respectively. For the first intermediate Lumi-R, the chromophore geometry is concluded to be the ZZEasa configuration. These finding imply that the primary step of the photoactivation of phytochrome is the Z/E isomerization of the C-D methine bridge double bond, whereas the single bond remains in the anti conformation. The subsequent transition to the physiologically active state Pfr includes a (partial) single bond rotation of the A-B methine bridge.     

STUDIES ON THE CAPACITY OF pR IN VITRO TO PHOTOCONVERT TO THE LONG-WVELENGTH Ffr-FORM. A SURVEY FO TEN PLANT SPECIES*

Studies on the capacity of Pr in uitro to photoconvert to the long wavelength in uioo-like Pfr form were performed with extracts from 10 species. Red irradiation, immediately after extraction of crude extracts from 9 species, photoconverted Pr to long-wavelength Pfr with an absorbance maximum around 735 nm. Red irradiation of soybean extracts, however, photoconverted Pr to short-wavelength Pfr, with an absorbance maximum at 725 nm. Red irradiation given later than 1.5-2 h after extraction, to extracts of oats, pea, cucumber, radish, sunflower and soybean, photoconverted Pr to a short-wavelength Pfr species with an absorbance maximum around 725 nm. In crude extracts of barley, corn, wheat and zucchini, red irradiation, even after a long dark-incubation period at 4°C of up to 48 h, photoconverted Pr to long-wavelength Pfr with an absorbance maximum around 735 nm. After incubation at 25°C for 3 h, however, Pr from barley also photoconverted to the short-wavelength form. It is suggested that in the group exemplified by oats, Pr rapidly undergoes an alteration following extraction, which results in the loss of the capacity of Pr to photoconvert to long-wavelength Pfr. In contrast, in extracts from the group exemplified by barley, Pr is much more stable and retains the capacity to photoconvert to long-wavelength Pfr for much longer periods.     

TWO SPECTROSCOPICALLY AND PHOTOCHEMICALLY DISTINGUISHABLE PHYTOCHROMES IN ETIOLATED SEEDLINGS OF MONOCOTS AND DICOTS *

Abstract Fluorescence (F) emission spectra of the red-absorbing phytochrome form (Pr) at 85 K, temperature dependence of the F intensity and the extent of the Pr F changes in the phototransformation of Pr into the first stable photoproduct (lumi-R) at 85 K (γ₁,) and into the far-red-absorbing form (Pfr) at 267 K (γ₂) were investigated in etiolated shoots and roots of monocots (oat, maize, rice) and dicots (pea, cress). These characteristics monotonously changed as a function of the phytochrome content, [Ptot]: with its decrease to 3-5% of the maximal values, the F spectrum shifts from 686 nm to 682 nm, its half-band width rises from 22 nm to ca 25 nm, temperature dependence of Pr F changes its character, γ¹, drops down from ca > 0.45 to ca 0.05-0.10 and γ₂ from 0.80–0.82 to ≤0.70. These data were interpreted in terms of two different phytochromes whose relative concentration varies with [Ptot]: (1) a longer wavelength type with the F maximum at 686 nm, low activation energy of the photoreaction (Ea ≤ 3–4 kj/ mol) and high extent of the phototransformation at 85 K (0.49 ± 0.03) and at 267 K (ca 0.85) (Pra); (2) a shorter wavelength type practically inactive at 85 K with F maximum at 682 nm, higher Ea (ca 35 kj/mol) and lower extent of the Pr & Pfr phototransformation (≤0.70) (Pri). [Pra] widely varies in different parts of the seedlings (up to 100 times) and Pra dominates when [Ptot] is high. The [Pri] is much more constant (variations, <10 times), and it becomes the major one when [Ptot] drops down. The two species are likely to belong to the labile (type 1) and stable pools of pigment and not to be connected with the localization of the pigment in the cell since red-far-red preillu-mination, which is believed to bring about sequestering of the pigment, does not change their relative concentration and properties.     

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.     

EVIDENCE FOR THE EXISTENCE OF MEMBRANE-ASSOCIATED PHYTOCHROME IN THE CELL

Abstract Comparative fluorescence and photochemical studies of phytochrome in etiolated seedlings of maize and in soluble and membrane-containing fractions isolated from them were camed out. The membrane fractions prepared in the absence of Mg²⁺ from etiolated coleoptiles contained 13% of total photoreversible phytochrome, which was readily solubilized by mild detergents. Its molecular size was indistinguishable from soluble phytochrome and equal to nondegraded maize phytochrome. Low-temperature fluorescence studies with intact tissue found that the position of the emission maximum at 85 K (λ_max) and the extent of the phototransformation of the red-absorbing form (Pr) into the first stable photoproduct, lumi-R, at 85 K (γ₁), varied in different parts of etiolated seedlings: λ_max and γ₁ reached their maximum values in the tips of coleoptiles and roots, 686 nm and 0.30–0.40, whereas the lowest values, 682 nm and ca 0.05, were observed in the root base. These parameters correlated well with those obtained for the pigment in the soluble and membrane-containing fractions: 684 and 680 nm, and 0.33 and 0.06, respectively. The extent of the Pr phototransformation into the far red-absorbing form (Pfr) (γ₂) did not differ much: values of 0.80–0.85 and 0.70–0.75 correlated with the high and low values of γ₁. These variations of the parameters were interpreted in agreement with our previous observations in terms of two phytochrome A species whose relative concentrations vary depending on the experimental conditions—the longer wavelength bulk light-labile species with high γ₁ (Pr″), and the shorter wavelength minor light-stable species with low γ₁ (Pr″). Close similarity between Pr’and the soluble phytochrome and between Pr″ and the membrane-bound phytochrome points to the possible origin of the native Pr’and PrPrime; species, thus providing evidence for the existence of membrane-bound pigment in the cell.