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.     

SPECTRAL PROPERTIES,PHOTOTRANSFORMATION KINETICS AND PELLETABILITY OF PHYTOCHROME IN Pharbitis nil Chois*

Abstract— An examination has been made of the involvement of phytochrome in the circadian rhythm of flowering in Pharbitis nil Chois. The peak position of P_fr absorption changes with time after a red light pulse. The shortest absorption wavelength of P_fr occurs at the same time as flowering is inhibited by red light in dark grown, red light pretreated plants. Pelletable and supernatant phytochrome show a similar trend with lowest values found at the time of flower inhibition. Neither phototransformation kinetics nor intermediates of phytochrome which accumulate in white light show such a relationship to the circadian rhythm found in flowering of dark grown P. nil.     

KINETICS OF PHYTOCHROME PELLETABILITY*

Abstract— The pelletability of P_r from maize coleoptiles was studied as a function of the delay time between a red and a far-red light pulse given in vivo. The obtained curve can be resolved into three parts. The two slowest reactions have half lives of 40 s and 3.6 min at 0°C. Furthermore, a break in the Arrhenius plot from the slowest reaction of the curve indicates that either the phytochrome “receptor” or the phytochrome molecule itself undergoes a jump in the Arrhenius activation energy at 20°C. These data are in good agreement with kinetic studies of phytochrome pelletability also discussed in this paper.     

PHOTOTRANSFORMATIONS OF PHYTOCHROME: THE CHARACTERIZATION OF LUMI-F AND META-Fa

Abstract— …Phototransformations of the red/far red reversible plant pigment phytochrome involve several intermediates. At 77K, lumi-F , the initial product of phototransformation of the far red absorbing form P _fr and some of its relaxation products are shown to undergo further phototransformations. Lumi-F has an absorption maximum in the region 690–730nm. The product, giving rise to a maximum in the difference spectra at 650nm, formerly thought to be lumi-F , is now believed to represent one of its relaxation products. The nature of the reactions connecting these various intermediates are discussed.     

ACTION SPECTRA OF PHYTOCHROME IN VIVO*

Abstract— A method is described to determine spectral properties of phytochrome in vivo. For photochrome in 7-day-old dark-grown Cucurbita pepo L. seedlings the mole fraction of the far-red-absorbing form (P_fr) present at photoequilibrium at 664 nm was found to be 0.76 ± 0.02 in vivo. Based on reflectance measurements, the photon fluence rate just below the surface of the cotyledons was calculated. Local rates of photoconversion for known local fluence rates were measured across cotyledons after non-saturating irradiations with wavelengths between 544 and 781 nm and in situ molar photoconversion coefficients were obtained. In contrast to purified oat phytochrome, the in situ molar photoconversion coefficients for P_fr show a strong shoulder between 660 and 700 nm. The maximum of P_fr absorption is at 726 nm. An isosbestic point of phytochrome is found at 686 nm. The mole fraction of P_fr present at photoequilibrium with 686 nm light is 0.58. The ratio of photoconversion quantum yields (that for P_r→ P_fr divided by that for P_fr→ P_r) is 1.38 ± 0.06.     

ANALYTICAL CHARACTERIZATION OF SPECTRAL ACTINIC FLUX and SPECTRAL IRRADIANCE IN THE MIDDLE ULTRAVIOLET

Abstract— We present an analytic characterization of upward and downward diffuse spectral irradiance for the wavelength range 280–380 nm, solar zenith angle range from 0 to 86, altitude range from 0 to 5 km and for non-zero surface albedo. This work is a modification and extension of the previous work of Green, Cross and Smith based upon the radiative transfer calculations of Braslau, Dave and Halpern. Guided by these irradiance systematics we develop an analytic characterization of diffuse spectral scalar irradiance or actinic flux also broken down into upward and downward components for the above wavelengths, solar zenith angles and altitudes, for non-zero surface albedo utilizing the actinic flux calculations of Peterson.     

苝四羧酸多联体化合物的合成及其光谱特性研究

本文合成了8个 四羧酸多联体化合物;测定了这些化合物的吸收光谱、荧光光谱。研究了由供电子分子键连的多联体分子的光谱特性及其分子体系内稳态荧光猝灭机理。首次从吸收光谱和荧光光谱上分析和考察了四羧酸多联体化合物分子体系内的光致电子转移反应。     

FLUORESCENCE QUENCHING OF ALLOPHYCOCYANIN AND PHYTOCHROME BY ESTRIOL*

Abstract— At 293 K the long-wavelength absorption and emission band of 1.4 μM allophycocyanin is decreased by estriol (Δ¹-³-⁵⁽¹⁰⁾-estratriene-3,16α, 17β-trio!) in the range 0.8-6.6 μM in the presence of 11% alcohol (vol/vol). The binding of estriol is shown to be of high affinity, 1:1 with allophycocyanin. The free energy of this binding process (ΔG^°) is -33.6 kJ mol' and single binding site dissociation constant (K_D) 1.0 ×10–6M. Estriol at 21 μM effectively quenches the fluorescence of 1.4 M large molecular weight phytochrome in its red absorbing form at 77 K while having little or no effect on the phototransformation difference spectrum at 293 K.     

IMMUNOBLOT ANALYSIS OF CROSS-REACTIVITY OF MONOCLONAL ANTIBODIES AGAINST OAT PHYTOCHROME WITH PHYTOCHROME FROM SEVERAL PLANT SPECIES*

Abstract— Eighteen monoclonal antibodies (mAbs) have been produced against partially degraded phytochrome from A vena sativa cv. Trafalgar. Several of the mAbs are capable of recognising the antigen on immunoblots following sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) and protein electroblotting. Six of these mAbs were screened for the ability to recognise electroblotted phytochrome from eight plant species. Particularly amongst the monocot species tested, but also amongst the dicots, the mAbs showed extensive cross-reactivity. The results suggest that there are perhaps several conserved antigenic sites on the phytochrome molecule.     

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.     

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 IN GREEN-TISSUE: PARTIAL PURIFICATION and CHARACTERIZATION OF THE 118-KILODALTON PHYTOCHROME SPECIES FROM LIGHT-GROWN Avena sativa L.*

The predominant, immunochemically-detectable phytochrome polypeptide rapidly extracted directly into boiling sodium dodecyl sulfate-containing buffer from fresh or freeze-dried green Avena tissue has an apparent molecular mass of 118 kilodaltons (kDa). This result indicates that the 118-kDa phytochrome species obtained from green Avena by extraction and rapid processing under non-denaturing conditions in previous studies was not derived by partial proteolysis of a larger polypeptide present in the cell. Additional data do, however, demonstrate the presence in green tissue homogenates of proteolytic activity that can cause a = 6-kDa reduction in apparent molecular mass and a blue-shift in the P_fr absorbance maximum of phytochrome during handling. This proteolytic activity contrasts with that previously encountered in etiolated tissue in that it is not inhibited by phenylmethylsulfonyl fluoride, but is inhibited by iodoacetamide and leupeptin. This result indicates that the activity is associated with a thiol-like protease. A partial purification procedure that incorporates the use of iodacetamide and a novel chromatographic step is described for green-tissue phytochrome. This procedure provides 50% recovery with a 90-fold enrichment of phytochrome relative to the initial extract in which the chromoprotein is 0.003% of the total soluble protein. The final fraction is apparently free of proteolytic activity. Immunoblot analysis of this fraction demonstrates that the predominant immunoreactive band has a monomeric molecular mass of 118 kDa. Comigration of this band with a band exhibiting zinc-induced fluorescence on blots of the partially purified preparations verifies that the 118-kDa species is the principal tetrapyrrole-bearing polypeptide present. Spectral properties of the final fraction are identical to those published for crude green-tissue extracts, indicating the stability of the molecule's spectral properties throughout the procedure. Size exclusion chromatography under nondenaturing conditions shows that the 118-kDa phytochrome species from green tissue comigrates with the dimeric, etiolated-tissue molecule, and is therefore suggestive of similar quaternary structure. Together these data reinforce previous conclusions that the predominant phytochrome molecule present in the living cells of green tissue is resolvable as a 118-kDa species, distinct from the well-characterized 124-kDa molecule from etiolated tissue (Tokuhisa et al., 1985, Planta 164, 321–332), and indicate that the partial purification protocol described here sustains the green-tissue phytochrome in its native state throughout the procedure.     

THE BLUE ANOMALOUS EMISSION OF LARGE AND SMALL PHYTOCHROME*

–Small and immunoaffinity-purified large phytochrome (P_r and P_fr) show a so-called anomalous fluorescence with λ^em_max= 470 and 440 nm, respectively, when irradiated within the blue absorption band. Model studies indicate that this emission arises from a dipyrromethenone partial structure produced by a nucleophilic addition to the central carbon C-10 of the bilindione chromophore. The anomalous emitter of phytochrome is thus similar to one bilirubin conformer which has previously been found to contribute to the absorption and emission of the bile pigment.     

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.     

PHOTOCHEMICAL CHARACTERIZATION OF COVALENTLY-LINKED PORPHYRIN-QUINONE COMPLEXES*

Abstract— The fluorescence properties of a covalently-linked porphyrin-quinone complex and its zinc derivative were studied in a variety of organic solvents. The kinetics of fluorescence decay for both the quinone and hydroquinone oxidation states were measured in acetonitrile, dichloromethane, dimethyl-formamide, and pentane. The fluorescence yield and kinetics of decay at room temperature were little affected in the porphyrin or zinc porphyrin complexes when the attached quinone was reduced. However, for these complexes the fluorescence yield and lifetimes were both substantially decreased in acetonitrile and dichloromethane when the quinone was in its oxidized state. These latter decay kinetics were not explainable by a process having a single exponential decay. On the other hand, little fluorescence quenching or lifetime shortening was observed in dimethylformamide or pentane, indicating unique solvent dependencies for the quenching process. Evidence was obtained for photoproduced charge separation from EPR measurements on the covalently-linked zinc porphyrin-quinone complex. The EPR data showed equivalent concentrations of a Zn porphyrin cation radical and a benzoquinone anion radical in acetonitrile or dichloromethane at both room temperature and 77 K. The charge separated state rapidly decayed at room temperature (in sub-millisecond times) but was quite stable at 77 K. It is concluded that light-induced charge separation in acetonitrile and dichloromethane at room temperature may occur from the excited singlet state with a high quantum efficiency. A photoproduced charge separated state also occurred when the covalently-linked complexes were incorporated into egg yolk phosphatidylcholine liposomes. The quantum yield for radical formation in this latter system was 0.1 and the lifetimes of the radical species formed were many minutes.     

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.     

LIGHT PERCEPTION BY PHYTOCHROME in vivo*

The rate of phytochrome photoconversion in cotyledons and hypocotyls of dark-grown seedlings of Cucurbita pepo varies significantly depending on the characteristics of the light field and the position of the sample in the light field. The overall pattern of the effects of light reflectance conditions and angle of incidence of light on phytochrome photoconversion in vivo is predictable and consistent with the results of physiological studies; however, the extent of the effects is much more difficult to predict with a satisfactory degree of approximation. The differences between phytochrome photoconversion kinetics measured in vivo and those predicted from photochemical parameters of purified phytochrome are rather large and suggest that the use of predictions as estimates of the state of phytochrome in vivo may be subject to significant limitations.