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.     

EXTENSION GROWTH AND ANTHOCYANIN RESPONSES OF PHOTOMORPHOGENIC TOMATO MUTANTS TO CHANGES IN THE PHYTOCHROME: PHOTOEQUILIBRIUM DURING THE DAILY PHOTOPERIOD

Four genotypes of tomato (Lycopersicon esculentum Mill.) in the genetic background ‘Aha Craig’ were used: an aurea (au) mutant, deficient in the bulk light-labile phytochrome pool; a high pigment (hp) mutant, showing exaggerated phytochrome responses at the time of de-etiolation; the au,hp double mutant and the isogenic wild type (WT). A dramatic increase in plant height resulting from an increase in the length of all internodes for each of the genotypes studied was observed upon reduction of the red light: far-red light photon ratio (R:FR) from 6.90 to 0.13 by addition of FR for the whole photoperiod. A concomitant increase in leaf length was also observed. Since au and au,hp mutants, deficient in the bulk light-labile phytochrome pool, respond to this reduction in the R:FR these data demonstrate that the phytochrome pool that mediates this response is present and fully functional. Anthocyanin was detectable in the comparably developed young growing leaves of the WT and hp mutant under the high R:FR, but not in the au and au,hp mutants, suggesting that the potential for anthocyanin synthesis is correlated with the presence of the bulk light-labile phytochrome pool. The kinetics of anthocyanin decrease in the young growing leaves were investigated in the hp mutant and the results suggest a very rapid cessation of flavonoid biosynthesis upon reduction of the R:FR. The functions of different phytochrome types are discussed.     

KINETIC STUDIES TO INTERPRET 'HIGH-ENERGY PHENOMENA' OF PHOTOMORPHOGENESISL ON THE BASIS OF PHYTOCHROME

Abstract— Kinetic studies with the mustard seedling ( Sinapis alba L.) support the hypothesis that the so-called 'high energy reaction' of photomorphogenesis can be understood solely on the basis of phytochrome. Light-induced anthocyanin synthesis (a typical 'positive' photoresponse⁽¹⁾ and light dependent inhibition of hypocotyl lengthening (a typical 'negative' photoresponse⁽¹⁾) have been investigated. In order to explain the experimental data we have to assume that there are two different types of phytochrome 730 which differ greatly as far as their resistance to irreversible destruction is concerned. The existence of these two different types of phytochrome 730 has already been postulated on the basis of spectrophotometric measurements in vivo .⁽²⁾     

A MODEL FOR THE MOLECULAR STRUCTURE AND ORIENTATION OF THE CHROMOPHORE IN A DIMERIC PHYTOCHROME MOLECULE*

A model for the molecular structure and orientation of red-light absorbing form of phytochrome (P,) chromophores in a dimeric molecular model of P_r is proposed. A chromophore model with probable molecular structures was generated to reproduce the absorption spectrum produced by its π-electron conjugating system. The model has C₅-Z, syn, C₁₀-E, anti and C₁₅-Z, syn configurations and a protonation at a C-ring nitrogen. Orientation of the chromophore model in the dimeric phytochrome molecular was analyzed by displaying the atoms of the chromophore, the coordinates of which were converted into those with respect to the molecular axes to the dimeric molecule, on a 3-D graphic workstation. The conversions were performed by using the azimuthal angles between the Z axis of the dimeric molecule (axis of 2-fold rotational symmetry) and the dipole moments of the electronic transition at the blue- (384 nm) and red- (667 nm) absorbing bands of the chromophore, which were calculated as 55.5° and 59.3°, respectively, based on linear dichroism of the oriented phytochrome molecules. The result demonstrates that the long axis of the P, chromophore lies almost parallel to the Y axis of the molecular model, and that the tetrapyrrolic chromophore is well contained within the flat chromophoric domain without protruding from it, a configuration that assures that the chromophore is protected against aqueous environments. The model may explain the rotation angle of the transition moment of the red-absorbing band, induced by the phototransformation from P_r to P_rr which we measured as smaller than that measured in nonoriented preparations by a photoselection technique. The model also suggests a molecular basis for the polarotropic response of phytochrome.     

STRUCTURAL STUDIES ON THE PHOTORECEPTOR PHYTOCHROME: REEVALUATION OF THE EPITOPE FOR MONOCLONAL ANTIBODY Z-3B1

The photoreceptor phytochrome is widely distributed in the plant kingdom from angiosperms to ferns, mosses and algae. The epitope for the monoclonal antibody Z-3B1 which exhibits wide-ranging cross-reactivity with phytochromes from higher and lower plants was mapped by the combination of several methods: by Western blot with proteolytic fragments of known localization, by sequence comparison of phytochromes from various plants, and by production of overlapping fusion proteins. The only sequence which is common to all positively-reacting fusion proteins is the sequence A-830 to R-859. This sequence must contain the Z-3B1 epitope. The best candidate is suggested to be the T-cell antigenic sequence K-Y-V/I-E-A/C-L-L-T (= K-848 to T-855). The significance of the highly conserved epitope in all phytochromes is discussed.     

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.     

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.     

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.     

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.     

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.     

A PHYTOCHROME PHOTOTRANSFORMATION STUDY USING TWO-LASER/TWO-COLOR FLASH PHOTOLYSIS: ANALYSIS OF THE DECAY MECHANISM OF I700*

The mechanism of I₇₀₀ decay, representing an early event in the phytochrome P_r→ P_fr phototransformation, was reanalyzed in the microsecond range by conventional laser flash photolysis as well as by two-laser/two-color flash photolysis. Three kinetic models that might describe the I₇₀₀ decay mechanism following P_r excitation were considered: a parallel, a sequential, and an equilibrium model. These models were used to mathematically simulate both the one- and two-laser flash experiments in an effort to select the model best describing the I₇₀₀ decay. The sequential model could be excluded already on the basis of the one-laser flash photolysis results alone. Discussion of the two-laser/two-color flash rcsults in the context of the equilibrium and the parallel models is presented.     

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.     

有机太阳能电池的染料结构与光电性能关系的研究

本文用自动测试系统探讨了菁染料和方酸染料的化学性质、结构与其光伏效应之间的关系, 从理论上讨论了载流子光致发生机理。发现在给电子体-受电子体发色团染料中, 光伏效应有显著的取代基效应, 同时观察到量子效率随给电子杂环的碱性及分子离子势等有一定变化规律。讨论了方酸染料的聚集态。