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.     

FLUORESCENCE OF PHYTOCHROME IN THE CELLS OF DARK-GROWN PLANTS AND ITS CONNECTION WITH THE PHOTOTRANSFORMATIONS OF THE PIGMENT

Abstract Fluorescence of phytochrome is found in the cells of etiolated monocotyledonous and dicotyledonous plants. The red light-absorbing form of phytochrome (P_r) fluoresces at 77 K with a yield 0.3±0.1 and maxima at 672–673 nm and 684–686 nm in the excitation and emission spectra, respectively. The emission is characterized by the sharp temperature dependence of its intensity, its high (～ 40%) polarization, and the violation of the mirror symmetry rule. Connection of the fluorescence with P_r photoreactions is followed in the interval 77–293 K. A P, photoproduct, lumi-R, is fluorescent with maxima at 696 nm and 705 nm in the excitation and emission spectra; the far-red light absorbing form of phytochrome (P_fr) is practically nonfluorescent. Three isochromic emitting P_r species are present differing in their photochemical properties: P_r1 and P_r2 which phototransform irreversibly and reversibly at T 170 K into lumi-R, and lumi-R₂, respectively, and P_r3 which undergoes photoconversion only at T > 240 K. The activation energies of P_r2 and P_r3 photoreactions are evaluated to be 2.9–3.3 kJ/mol and 26 kJ/mol. Complex dynamics of changes of P_r fluorescence and of the extent of its decrease in the photoconversion P_r? P_fr in germinating pea and bean seeds suggests the existence of two P_r pools one of which is incapable of P_r? P_fr phototransformation. Thus, the developed fluorescent method of phytochrome assay and investigation in the cell revealing multiplicity of phytochrome states in vivo proves to be very sensitive (about 1 ng) and informative.     

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.     

PHYTOCHROME CONTROL OF THE DEVELOPMENT OF PHOTOPHOSPHORYLATION

In the cotyledons of the mustard (Sinapis ah L.) seedling the development of the capacity for photophosphorylation is strongly influenced by pretreatment of the seedling with red light pulses. The red light acts through phytochrome. After a red light pretreatment the capacity for photophosphorylation increases linearly with the chlorophyll content, at least up to 30 min after the onset of continuous white light. It is proposed that the reaction chain required for photophosphorylation is completed under the influence of phytochrome even in the absence of chlorophyll. As soon as chlorophyll becomes available photophosphorylation functions instantaneously. Without a red light pretreatment there is a lag of more than 15 min before photophosphorylation becomes detectable after the onset of continuous white light even though chlorophyll a is available. Although phytochrome strongly influences the rate of chlorophyll accumulation as well it is improbable that the control by phytochrome of development of photophosphorylation and of chlorophyll accumulation are causally connected.     

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.     

THE TEMPERATURE DEPENDENCE OF PHYTOCHROME TRANSFORMATIONS

Abstract— The kinetics of phytochrome transformation were examined over a 30°C temperature range (+5° to–25°C) in 75% glycerol. Two new intermediate reaction stages are described for the transformation of the red-absorbing form, P _r to the far-red absorbing form, P _fr The free energies, enthalpies and entropies of activation were obtained for five of the six reaction stages observed in the transformation of P _r to P _fr and for the two reaction stages observed in the reverse process. All exhibited positive entropies of activation with the maximum being 25 entropy units. The results suggest that the phototransformations between P _r and P _fr consist of relaxation processes beginning with the intermediate produced immediately upon absorption of a quantum of light and ending with either P _r or P _fr.     

SPECTRAL CHARACTERIZATION OF HIGH-MOLECULAR-WEIGHT PHYTOCHROME*

Abstract— Previous information about the spectral and photochemical properties of phytochrome in vitro has apparently been determined in large part with chromopeptides derived from the native molecule by proteolysis. Characterization of high-mol-wt phytochrome in vitro has led to the observation that the far-red-absorbing form (Pfr) may undergo relatively large and reversible changes in far-red extinction. Phytochrome preparations which exhibit reduced far-red extinction as Pfr also exhibit a rapid reappearance of red absorbance after discontinuing the red illumination used to establish photostationary equilibrium. This rapid change in the red spectral region is not accompanied by any equivalent absorbance change in the far-red. The molecular basis for these newly reported spectral properties is not known. However, both properties may be eliminated by the addition of either 2-mercaptoethanol or ethylenediaminetetraacetic acid.     

THE FUNCTION OF PHYTOCHROME IN THE NATURAL ENVIRONMENT—III. MEASUREMENT AND CALCULATION OF PHYTOCHROME PHOTOEQUILIBRIA

Abstract— Phytochrome photoequilibria have been measured in dark-grown Phaseolus uulgaris L . and Cucurbita pepo I . hypocotyl hooks which had been exposed to various natural and artificial radiation sources. Mean phytochrome photoequilibria ( φ ) varied from 0.20 within a wheat canopy to 0.54 above, although lower values were occasionally observed in densely shaded areas. Greater variation in phyto chrome photoequilibria and lower levels of P_fr were recorded within a sugar beet canopy. The range of photoequilibria was φ= 0.04 in dense shade to φ= 0.54 above the canopy. Photoequilibrium was achieved within 5 s in mid-day sunlight and approximately 30 s in dense canopy shade.
A close correlation was found between φ and the ratio of the quantum flux in the red and far-red wavelength bands (ζ) in broad spectrum (400–800 nm) radiation. This relationship allows direct prediction of φ from a knowledge of ζ. Phytochrome showed greatest sensitivity to spectral changes in the range ζ= zero to ζ= 1.0, which is the range found in the natural environment.
The observations provide support for the hypothesis that phytochrome is involved in the detection of shading by plants.     

CONTROL OF CHLOROPHYLL b BIOSYNTHESIS BY PHYTOCHROME

In the mustard seedling cotyledons, chlorophyll b appears from the very beginning in white light provided that a red light pulse pretreatment was given 12 h prior to the onset of white light. The red light pulses act through phytochrome. Without pretreatment no chlorophyll b is detectable at least during the first 60 min after the onset of white light (25°C). Biogenesis of chlorophyll b specifically depends on the action of phytochrome during the pre-steady state period as well as during the steady state period of chlorophyll accumulation. In light pulse experiments, it was found that formation of chlorophyll b takes place stoichiometrically at the cost of chlorophyll(ide) a.     

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.     

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.     

ANALYSIS OF THE BINDING OF PHYTOCHROME TO PARTICULATE FRACTIONS

Abstract— The binding of phytochrome to receptor sites in a particulate fraction of maize coleoptiles has been studied as a function of the level of far-red-absorbing phytochrome (P_fr) offered in vivo and in vitro. Evidence is presented that the binding is cooperative. The degree of cooperativity expressed by the Hill coefficient of the binding function is the same (1–6) both in vivo and in vitro , whereas the Hill coefficient of the state function in vivo is significantly higher (2-1). The highest Hill coefficient (3–5) was found for the in vitro binding function in squash hooks.     

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.     

THE ROLE OF HYDRATION IN THE PHOTOTRANSFORMATION OF PHYTOCHROME

Abstract— Changes in the visible absorption spectrum and in phototransformation of phytochrome have been found to be associated with dehydration and rehydration. These spectral properties explain the previously reported rapid appearance of phytochrome in imbibing seeds. No evidence was found for the orientation of the phytochrome chromophore in gelatin films or in preparations subjected to a shearing force.     

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.