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.     

The Complexity of the Pr to Pfr Phototransformation Kinetics Is an Intrinsic Property of Native Phytochrome*

Several possible origins of the complex phytochrome red to far-red light-absorbing phytochrome (P_r P_fr) phototransformation kinetics in the nanosecond-to-second time range have been examined. Heterogeneity based on protein sequence is ruled out as an origin of the multi-component kinetics because recombinant 124 kDa oat phytochrome A apoprotein reconstituted with phytochro-mobilin and the native protein are very similar in this regard throughout this time range. The P_r forms of native 124 kDa oat phytochrome A and of a homogeneous recombinant 65 kDa chromoprotein fragment exhibit thermochromic properties interpreted as arising in each case from the presence of two P_r species in thermal equilibrium. They exhibit identical photochemical properties. The complex kinetics therefore cannot result from P_r heterogeneity either. Thus, the presence of two P_r forms in equilibrium (P_r,₆₇₅ and P_r,₆₅₅) and the complex multiex-ponential P_rP_fr phototransformation kinetics observed in all time ranges are intrinsic properties of the homogeneous holoprotein of oat phytochrome A.     

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.     

The molecular topography of phytochrome: chromophore and apoprotein

Phytochrome serves as the photochromic receptor for a number of morphogenic and developmental responses to red light in higher plants. The photoreversible phototransformation of 124 kDa oat phytochrome involves several structural changes in the chromophore and the apoprotein, including a configurational/conformational isomerization and secondary/tertiary structural changes respectively. For example, there appears to be a specific interaction between the chromophore and the amino terminus segment in the Pfr form of phytochrome, which results in a photoreversible peptide folding of the amino terminus peptide chain. Other structural changes also accompany the phototransformation, as has been probed by peptide mapping, phosphorylation, and monoclonal antibodies.     

A SIMPLE AND IMPROVED METHOD OF ISOLATION AND PURIFICATION FOR NATIVE OAT PHYTOCHROME

Abstract— A simple procedure for the isolation and purification of 124 kDa phytochrome (phyA) from etiolated Avena seedlings has been developed employing ammonium sulfate back-extraction. After solubilization of the ammonium sulfate precipitate (250 g/L) an additional ammonium sulfate fractionation with 17 g per 100 mL rather than column chromatography was performed. After several steps of the "washing-out" procedure with 100 mM phosphate buffer, phytochrome was solubilized in 10 m M phosphate buffer. The resulting phytochrome had a specific absorbance ratio (SAR = A₆₆₆/A₂₈o) ranging from 0.60 to 0.85. These values are equivalent to those of phytochrome preparations after hydroxyla-patite chromatography-ammonium sulfate back-extraction. The total isolation-purification time was 8 h and yield of the chromoprotein was 50% higher than the yield using conventional techniques. The phytochrome preparation, after application to a Toyopearl HW-65S gel filtration column, produced very pure 124 kDa phyA with a specific absorbance ratio greater than 1.00. The spectral characteristics are identical to those described for the best of the highly purified native chromoprotein preparations.     

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.     

The photoreactions of recombinant phytochrome from the cyanobacterium Synechocystis: a low-temperature UV-Vis and FT-IR spectroscopic study

The interconvertible photoreactions of recombinant phytochrome from Synechocystis reconstituted with phycocyanobilin were investigated by light-induced optical and Fourier-transform infrared (FT-IR) difference spectroscopy at low temperatures for the first time. The photochemistry was found to be deferred below -100 degrees C for the transformation of red-absorbing form of phytochrome (Pr)-->far-red-absorbing form of phytochrome (Pfr), and no formation of an intermediate similar to the photoproduct of phytochrome A obtained at -140 degrees C (lumi-R) was observed. Two intermediates could be stabilized below -40 degrees C and between -40 and -20 degrees C, and were denoted as meta-Ra and meta-Rc, respectively. Above -20 degrees C Pfr was obtained. In the reverse reaction two intermediates could be stabilized below -60 degrees C (lumi-F) and between -60 and -40 degrees C (meta-F). The FT-IR difference spectra of the late Pr-->Pfr photoreaction show great similarities to the spectra obtained from oat phytochrome A suggesting similar conformation of the chromophore and interactions with its protein environment, whereas deviations in the spectra of meta-Ra were observed. A large band around 1700 cm-1 in the difference spectra between the intermediates and Pr which is tentatively assigned to the C19=O group of the prosthetic group indicates the Z,E isomerization around the C15=C16-methine bridge of the chromophore during the formation of meta-Ra. In the difference spectra of the parent states only small differences are observed in this region suggesting that the frequency of the carbonyl group is similar in Pr and Pfr. Since the FT-IR difference spectra between lumi-F and Pfr show great similarities to the spectra of the parent states, it is assumed that during the formation of lumi-F the chromophore largely returns into the primary Pr conformation. The FT-IR spectra recorded in a medium of 2H2O generally show a downshift of the significant bands due to the isotope effect. The appearance of a characteristic band around 935 cm-1 in all 2H2O spectra suggests an assignment to an N-2H bending vibration of the chromophore.     

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.     

The photoreactions of recombinant phytochrome CphA from the cyanobacterium Calothrix PCC7601: a low-temperature UV-Vis and FTIR study

The photoreactions of recombinant phytochrome CphA from cyanobacterium Calothrix sp. PCC7601 reconstituted with phycocyanobilin were investigated using UV–Vis and Fourier transform infrared (FTIR) difference spectroscopy, stabilizing intermediates at low temperature. The yield of the forward reaction strongly depends on temperature, unlike the backward reaction. Because of the very fast thermal relaxation processes in the Pr to Pfr pathway, no pure difference spectra of the Pr photoconversion products could be directly measured. Thus, the contribution of the Pfr:Pr pathway was taken into account by applying an appropriate correction procedure both in the UV–Vis and FTIR experiments. Three intermediates have been trapped at −25, −45 and −120°C, which show the characteristic vibrational band pattern of the plant phytochrome phyA intermediates meta-Rc, meta-Ra and lumi-R, respectively. In the backward reaction, two intermediates corresponding to meta-F and lumi-F were trapped at −70 and −140°C, respectively. FTIR spectra of all intermediates, as well as of the Pfr state, show remarkable similarities with the corresponding spectra of Cph1 phytochrome from cyanobacterium Synechocystis and the 59 kDa N-terminal fragment of Cph1, and, albeit not so pronounced, also with plant phyA. The spectral similarities and differences between the various phytochromes are discussed in terms of structural changes of the chromophore and the chromophore–protein interactions.     

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.     

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.     

Phytochromes with noncovalently bound chromophores: the ability of apophytochromes to direct tetrapyrrole photoisomerization

Chromophore-apoprotein interactions were studied with recombinant apoproteins, oat phytochrome (phyA) and CphB of the cyanobacterium Calothrix PCC7601, which were both incubated with the bilin compounds biliverdin (BV) IXalpha, phycocyanobilin (PCB) and the 3'-methoxy derivative of PCB. Previously it was shown that CphB and its homolog in Calothrix, CphA, show strong sequence similarities with each other and with the phytochromes of higher and lower plants, despite the fact that CphB carries a leucine instead of a cysteine at the chromophore attachment position and thus holds the chromophore only noncovalently. CphA binds tetrapyrrole chromophores in a covalent, phytochrome-like manner. For both eyanobacterial phytochromes, red and far-red light-induced photochemistry has been reported. Thus, the role of the binding site of CphB in directing the photochemistry of noncovalently bound tetrapyrroles was analyzed in comparison with the apoprotein from phyA phytochrome. Both the aforementioned compounds, which were used as chromophores, are not able to form covalent bonds with a phytochrome-type apoprotein because of their chemical structure (vinyl group at position 3 or methoxy group at position 3'). The BV adducts of both apoproteins showed phytochrome-like photochemistry (formation of red and far-red-absorbing forms of phytochrome [P(r) and P(fr) forms]). However, incubation of the oat apophytochrome with BV primarily yields a 700 nm form from which the P(r)-P(fr) photochemistry can be initiated and to which the system relaxes in the dark after illumination. The results for CphB were compared with a CphB mutant where the chromophore-binding cysteine had been introduced, which, upon incubation with PCB, shows spectral properties nearly identical with its (covalently binding) CphA homolog. A comparison of the spectral properties (P(r) and P(fr) forms) of all the PCB- and BV-containing chromoproteins reveals that the binding site of the cyanobacterial apoprotein is better suited than the plant (oat) phytochrome to noncovalently incorporate the chromophore and to regulate its photochemistry. Our findings support the proposal that the recently identified phytochrome-like prokaryotic photoreceptors, which do not contain a covalently bound chromophore, may trigger a light-induced physiological response.     

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.     

PHOTOTRANSFORMATION KINETICS OF UNDEGRADED OAT AND PEA PHYTOCHROME INITIATED BY LASER FLASH EXCITATION OF THE RED-ABSORBING FORM

Abstract— Phototransformation at 2°C of the red-absorbing form of phytochrome (Pr) to the far-redabsorbing form (Pfr) was studied with both undegraded oat ( Avena sativa L., cv. Garry) and undergraded pea Pisum sativum L., cv. Alaska) phytochrome. Phototransformation was initiated by a 15-ns laser pulse with maximum emission near 600 nm and output power of 30 mJ. The first resolvable transformation intermediate exhibited relative to Pr a maximum absorbance increase near 700 nm and was fully present at the earliest time measured, which was 60 ns after the flash. This intermediate absorbance decayed by two reactions for oat phytochrome (half-lives of 11 and 140 μs assuming parallel reactions) and by three for pea phytochrome (half-lives of 14, 280 and 1600 μs assuming parallel reactions). The kinetics of the slowest reaction for pea phytochrome, however, might be somewhat distorted by an instrument artifact. The appearance of the far-red-absorbing phytochrome, as monitored by absorbance increase at 720 nm, occurred by at least two reactions for both oat (half-lives of 47 and 250 ms assuming parallel reactions) and pea (half-lives of 170 and 770 ms assuming parallel reactions) phytochrome. The possibility of slower reactions was not tested. Assays for possible proteolysis of the phytochrome samples studied here indicated that the presence of degraded phytochrome could not account for the observed multiphasic kinetics except possibly for one phase of the triphasic intermediate decay seen with pea phytochrome.     

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.     

EXPRESSION OF OAT phyA cDNA IN THE MOSS Ceratodon purpureus

The possibility of transforming Ceratodon purpureus protoplasts by PEG-mediated direct DNA uptake was tested. Transformation with a plasmid carrying a kanamycin-resistance gene resulted in kanamycin-resistant colonies of C. purpureus protonemata. A full-length cDNA clone coding for oat phyA phytochrome was isolated. The clone HM4.1 which is 3.7-kb long exhibits about 99% nucleotide sequence identity to the known phytochrome clone AP3. The expression of HM4.1 in C. purpureus protonemata was tested. A construct with the 35S-promotor and the structural gene of HM4.1 was cotransformed with the plasmid containing the kanamycin-resistance. Kanamycin-resistant colonies were tested for the presence of HM4.1 sequences in a genomic Southern experiment. Two out of 19 kanamycin-resistant colonies reacted positively with a HM4.1 specific probe. The expression of phyA in the positive colonies was examined with monoclonal antibodies specific for oat phytochrome. The Western blot experiment with protein extracts of the two positive colonies grown in the dark revealed clear signals at 124-kDa which were not detected in control plants. These data demonstrate the possibility of expressing oat phyA-apoprotein in C. purpureus protonemata. The transgenic moss protonemata did not show phenotypical alterations in response to the foreign phytochrome polypeptide; it is not known at the moment if the tetrapyrole chromophore is attached to the oat polypeptide in the protonemata or not.     

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.     

Time-resolved thermodynamic analysis of the oat phytochrome A phototransformation. A photothermal beam deflection study.

The time-resolved enthalpy and the structural volume changes after excitation of native oat phytochrome A were studied in the micro- to milliseconds range by photothermal beam deflection (PBD), a technique that follows the time-resolved refractive index changes upon decay of the excited species. The first set of intermediates, I700(1) and I700(2), stores ca 83% of the energy of the first excited state, in agreement with previous optoacoustic data, whereas the second set stores only ca 18%. The temperature dependence of the amplitudes ratio for the optical absorbances of the (I700(1) + I700(2)) intermediates set is explained on the basis of the thermochromic equilibrium between Pr,657 and Pr,672, which also is in line with the present PBD data. These data were best fitted with a parallel mechanism (with equal yield in each branch) for the production of the first set of intermediates, I700(1) and I700(2), as well as the second set of intermediates, Ibl1 and Ibl2. Thus, the final steps toward Pfr should be largely driven by positive entropic changes brought about by protein movements, in line with previous resonance Raman data. For the production of the first set of intermediates (I700(1) and I700(2)) an expansion of 18 +/- 13 mL mol-1 was determined, and a further expansion > or = 7 mL mol-1 was estimated for the decay from I700(1) to the set of Ibl intermediates, indicating that the far red-absorbing form of phytochrome (Pfr) has a larger volume than the red-absorbing form of phytochrome. This is in agreement with previous chromatographic and circular dichroism data according to which Pfr shows a larger volume and the chromophore shows a higher accessibility, respectively, in the Pfr state.     

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.     

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.