PHYTOCHROME-MEDIATED PHOTOTROPISM IN PROTONEMATA OF THE MOSS Ceratodon purpureus BRID

Abstract— Protonemata of the moss Ceratodon purpureus cultured in white light were transferred to darkness for 3 days and then used for phototropic experiments. Irradiation of the apical region of vertically position protonemata with small beams (0.2 mm) of red light induced a growth response towards the irradiated side (positive phototropism). The phototropic response showed irradiance dependence. The effect of red light was completely reversed by far-red light following red light irradiations, demonstrating that phytochrome was the photoreceptor pigment. Far-red light or UV-blue light had no influence on either bulging or phototropism. Experiments with linearly polarized red or far-red light showed a different dichroic distribution of phytochrome in its different forms, the red-absorbing form, P_r and the far-red-absorbing form, P_fr. Red light with a vibration plane parallel to the long axis of the filaments was most effective. The effectiveness of far-red light was expressed best when its vibration plane was 90° to the electrical vector of the inductive red light.     

INTRACELLULAR DICHROIC ORIENTATION OF THE BLUE LIGHT-ABSORBING PIGMENT AND THE BLUE-ABSORPTION BAND OF THE RED-ABSORBING FORM OF PHYTOCHROME RESPONSIBLE FOR PHOTOTROPISM OF THE FERN Adiantum PROTONEMATA

The intracellular localization and orientation of the receptors for the blue light-induced phototropism in the fern Adianrum protonemata, phytochrome and the blue light-absorbing pigment, were investigated by combining the techniques of cell centrifugation and of microbeam irradiation with linearly polarized light. The phototropic response was induced in the cells even after they had been centrifuged basipetally to spin down the endoplasm from the apical region. When a polarized blue microbeam was given to a flank of the apical region of the protonema, the phototropic response after compensation of phytochrome effect by far-red light was most effectively induced when the polarization plane was parallel to the long axis of the cell. If protonemata were pre-irradiated with blue and far-red light, the phototropic response was mediated through phytochrome alone. If such pre-irradiated protonemata were similarly irradiated with a polarized blue microbeam, polarized light vibrating parallel to the cell axis was again most effective in inducing the response. These results indicate that both the blue light-absorbing pigment and the phytochrome responsible for the blue light-induced phototropism in Adiantum are confined to the plasma membrane and/or the ectoplasm and that the transition moments of their blue-absorption bands are nearly parallel to the cell surface.     

LIGHT-INDUCED LINEAR DICHROISM IN PHOTOREVERSIBLY PHOTOCHROMIC SENSOR PIGMENTS–II. CHROMOPHORE ROTATION IN IMMOBILIZED PHYTOCHROME

-Large phytochrome immobilized via anti-phytochrome immunoglobulin bound to Sepharose beads was irradiated to saturation with unpolarized far-red light. The apparent absorbance level was recorded in a dual wavelength spectrophotometer with both measuring beams set to either 660 or 730 nm and polarized perpendicular to each other. The sample was then irradiated with red polarized light. The apparent change in absorbance obtained after this irradiation indicated that purified phytochrome could show linear dichroism. From the absorbance values obtained it was computed that the direction of the long-wavelength transition moment changes by either 32 or 148^o, when phytochrome is transformed from P_r to P_fr. Considering the model of Hahn and Song (1981) the latter value appears more likely. In light of these results, the conclusions drawn from in vivo experiments on action dichroism in Dryopteris (Etzold, 1965), Adiantum (Kadota et al., 1982) and Mougeoutia (Haupt. 1970), which point to a 90^o rotation. should be reconsidered.     

PHOTOCONVERSION OF PHYTOCHROME IN VIVO STUDIED BY DOUBLE FLASH IRRADIATION IN Mougeotia AND Avena

Abstract— The kinetics of phytochrome phototransformation from the red-absorbing form (P_r) to the far-red-absorbing form (P_fr) in vivo at 22°C were studied using a double flash apparatus with 1-ms flashes. Photoconversion by simultaneous flashes of red light saturates at a low P_fr level, indicating the possible attainment of a photoequilibrium between the excitation of P_r and the photoreversion of intermediates in the course of the I-ms flashes. At saturation energy, simultaneous flashes resulted in about 50% as much P_fr as was produced by saturating irradiation with 5 s red light. Intermediates of the phototransformation pathway were analysed by separating two red or a red and a far-red flash by variable dark intervals. In both plants phototransformation intermediates with half-lives < 1 ms occur, but they are too short-lived to characterize by our method. The subsequent intermediates have half-lives of about 7 ms and 150 ms in A vena , 2 ms and 10 ms in Mougeotia. The conversion from P_r to P_fr seems to be completed 1 s after the red flash in Avena. In the alga Mougeotia , P_fr formation seems to be finished within only 50 ms after the inducing red flash. The kinetics obtained from physiological and spectrophotometric experiments with Avena mesocotyls are almost identical. These observations indicate that the physiological response corresponds directly to the amount of P_fr produced and not to phototransformation intermediates or "cycling" between P_r and P_fr.     

COMPARATIVE PHOTOCHEMICAL ANALYSIS OF HIGHLY PURIFIED 124 KILODALTON OAT and RYE PHYTOCHROMES in vitro

Abstract A direct comparison of the photochemical interconversions between red (P_r-) and far-red (P_fr-) absorbing forms of highly-purified 124 kDa oat and rye phytochromes under identical experimental conditions was performed. In two different buffer systems at 5°C, the quantum yields for the P_r to P_tr and P_fr to P_r phototransformations under constant red and far-red illumination, φ _r and φ_fr respectively, were determined to be 0.152-0.154 and 0.060-0.065 for oat preparations and 0.172-0.174 and 0.074-0.078 for rye preparations. These values as well as the wavelength dependence of the photoequilibrium produced under continuous illumination throughout the visible and near-ultraviolet spectrum were based on the absorption spectra of the two phytochrome preparations and revised molar absorption coefficients. The molar absorption coefficients were estimated by quantitative amino acid analysis and shown to be identical for the two monocot phytochromes (i.e. 132 mM ⁻¹ cm⁻¹ at the red absorption maximum for the P_r form). Because these measurements were performed under identical experimental conditions, including buffer, temperature, light fluence rate, and instrumentation, the differences observed must reflect structural features inherent to the two different monocotyledonous phytochromes.     

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.     

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.     

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.     

INDEPENDENT EFFECTS OF PHYTOCHROME AND NITRATE ON NITRATE REDUCTASE AND NITRITE REDUCTASE ACTIVITIES IN MAIZE

Abstract— Involvement of phytochrome in the regulation of nitrate reductase (NR) and nitrite reductase (NIR) activities in excised, etiolated leaves of Zea mays (L.) variety 'Ganga-5' is demonstrated using low energy and high irradiance responses of phytochrome action. Photoreversibility by far-red light of red light stimulated increases in NR and NIR activities was lost by 2 h. Red light given to the leaves, when induction by NO^-₃, was saturated, further increased both enzyme activities. Even if red light was given 4–8 h before NO^-₃, it still increased both NR and NIR activities.     

PHYTOCHROME INTERMEDIATES IN VIVO– III KINETIC ANALYSIS OF INTERMEDIATE REACTIONS AT LOW TEMPERATURE

Abstract— Kinetic experiments have provided evidence for a series of light and dark reactions of phytochrome intermediates at low temperature in Pisum epicotyl tissue. A photoequilibrium exists between P_r and P₆₉₈, and between P_fr and P₆₅₀. A dark reversion of P₆₉₈P_r and P₆₅₀p_fr at –70°C has been demonstrated. When cooled to 70°C under incandescent light, most of the phytochrome in the tissue is driven into photochemically unreactive intermediates. About 2% of the phytochrome remains as weakly absorbing intermediates that form P_r and P_fr in darkness. A scheme is presented for phytochrome phototransformation in vivo.     

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.     

CONTROL BY PHYTOCHROME OF EXTENSION GROWTH and POLAROTROPISM IN CHLORONEMATA OF Funaria hygrometrica

Abstract— The photocontrol of extension growth and polarotropism has been investigated in chloronemata of the moss Funaria hygrometrica Hedw. When grown on a 12/12 h light/dark cycle, chloronemata show light-stimulated diurnal variations in elongation rate with no evidence of a circadian rhythm. Regulation of elongation by a low energy, photoreversible phytochrome mechanism was demonstrated by brief red (R) and far red (FR) irradiations given either at the end of the day (EOD) or 6 h later as a night break (NB). Night break irradiation with polarised R caused polarotropic reorientations of chloronemal growth also through a FR-reversible, low energy phytochrome mechanism. Such transformations of P_r to P_fr were accompanied by a 90° shift in the orientation of the phytochrome chromophore. Microbeam irradiation indicated that the phytochrome chromophores involved in both responses were predominantly, if not entirely, located in the tip of the apical chloronemal cell.     

DEPENDENCE OF Pfr/Ptot-RATIOS ON LIGHT QUALITY and LIGHT QUANTITY

Abstract— Not only the spectral distribution of the light source determines the relative proportion of phytochrome in the P_fr(P_r) form, the P_fr/P_tot-ratio also depends strongly on the fluence rate of the irradiation. This dependence has been observed in the cotyledons of etiolated mustard seedlings for blue light of fluence rates below 20 Wm_-2. It has also been observed for white light and seems to be a characteristic of the phytochrome system resulting from the involvement of phytochrome thermal reactions as well as P_r P_fr photoconversions. The fluence rate dependence of P_fr/P_tot-ratios can be used to analyze the characteristic transformations of the phytochrome system. Phototransformations together with a fast thermal transformation (τ_½⋍ 3min) are consistent with the results obtained for blue and white light.     

STIMULATION OF THE SHIBATA SHIFT BY PHOTOCHROME IN THE COTYLEDONS OF THE MUSTARD SEEDLING SINAPIS ALBA L.

Abstract— In the cotyledons of the mustard seedling Sinapis alba L. the duration of the Shibata shift can be greatly shortened by a pretreatment with light pulses prior to the protochlorophyllide– chloro-phyllide a photoconversion. It was shown that the light pulses act through photochrome (P _fr ). Since reversibility of a red light pulse induction by a far-red light pulse is rapidly lost (within 2 min) it is concluded that at least the initial action of P_fr occurs rapidly in this response. On the other hand, the effect of a red light pulse on the rate of protochlorophyll regeneration in the mustard seedling cotyledons is fully reversible by a far-red light pulse for more than 5 min. It is concluded that control of protochlorophyll regeneration and control of the Shibata shift by phytochrome cannot be consequences of the same initial action of P_fr Apparently P_fr controls both phenomena independently.     

LIGHT-INDUCED LINEAR ACTION DICHROISM IN PHOTOREVERSIBLY PHOTOCHROMIC SENSOR PIGMENTS—I. THEORY

Abstract— With a photoreversibly photochromic regulator pigment such as phytochrome, linear action dichroism could theoretically be obtained after photoselection even if the molecules are initially randomly oriented: If randomly oriented P_fr (fed-absorbing phytochrome) molecules are partially converted to P_fr (far-red absorbing phytochrome) molecules by plane-polarized red light, those molecules will preferentially be converted which have their 'red' transition moments nearly parallel to the electric vector of the red light. The effect of subsequent plane-polarized far-red light will depend on the plane of polarization. A general theory is developed for how this can be used to determine whether or not the transition moment changes direction during conversion. The pigment need not be isolated, since only physiological reactions (such as germination or chromatic adaptation) are measured.     

CIRCULAR DICHROISM OF THE PHYTOCHROME CHROMOPHORES IN NATIVE AND DENATURED STATE

Abstract— The circular dichroism spectra of oat phytochrome were recorded. Qualitatively, the same spectra were found for large (360 kilodaltons) and small (60 kilodaltons) phytochrome. Quantitative CD data were reported for small P_r and P_fr (photoequilibrium mixture with 20% P_r) in tris buffer (native state) and in acid urea (denatured state). Further, the CD spectra of a phytochromobilinpeptide in acid solution with and without urea were recorded. Differences between the data in native and denatured state are discussed.     

RESONANCE RAMAN SPECTRA OF THE Pr-FORM OF PHYTOCHROME

Abstract— Resonance Raman spectra of the P_r-form of oat phytochrome have been obtained at 77 K. Interference from phytochrome fluorescence is avoided by employing far-red 752 nm excitation. Vibrational assignments are suggested for the tetrapyrrole chromophore in phytochrome by comparison with previously published model compound spectra and by examining the characteristic shifts induced by deuteration of the pyrrole nitrogens. The lack of carbonyl intensity, the frequencies of the 1626 and 1644 cm^-1 C=C stretching modes, and the presence of an intense mode at 1326 cm^-1 are all consistent with a protonated structure for the tetrapyrrole chromophore in P_r. This suggests that the -50 nm red-shift of the protein-bound chromophore absorption compared to the chromophore in vitro is caused by protonation of the pyrrole nitrogen.     

PHYTOCHROME-MEDIATED CONTROL OF PROLAMELLAR BODY REORGANIZATION AND PLASTID SIZE IN MUSTARD COTYLEDONS

Abstract— The development of plastids in the palisade parenchyma cells of the cotyledons of mustard seedlings ( Sinapis alba L.) was studied by electron microscopy. In darkness the etioplasts undergo a sequence of morphogenic changes previously recognized in principle in bean and barley leaves, as summarized by Rosinski, J. and W. G. Rosen (1972) Quart. Rev. Biol. 47 , 160–190. From 12 to 36 h after sowing, an increase in the percentage of etioplast profiles with paracrystalline prolamellar bodies can be observed. Thereafter, the degree of organization and size of the prolamellar bodies decrease. 60 h after sowing, the etioplasts show only remnants of prolamellar bodies with irregularly spaced tubules. Continuous far-red light, which is considered to operate via phytochrome, counteracts the decay of organization of the prolamellar body and strongly increases the size of the plastids. The effect of continuous far-red light (onset of light 36 h after sowing) can be substituted by 12 h of far-red light given between 36 and 48 h after sowing. It is shown with red and far-red light pulses that the morphogenic effect of long-term far-red light on plastid size and appearance of the prolamellar body is exclusively due to phytochrome (P_fr). Changes by light in the amounts of protochlorophyll(ide) or chlorophyll(ide) do not affect these results. The action of P_fr on the structure of the prolamellar body is a relatively fast process, occurring within 3 h. Formation of thylakoids does not seem to be under phytochrome control. Rather, this response seems to be related to the protochlorophyll(ide)→ chlorophyll(ide) a transformation.     

MODE OF COACTION OF PHYTOCHROME AND BLUE LIGHT PHOTORECEPTOR IN CONTROL OF HYPOCOTYL ELONGATION

Abstract The rate of hypocotyl longitudinal growth in seedlings of Sesamum indicum L. is strongly inhibited by continuous blue light (cBL)† and slightly by continuous far-red light while continuous red light (cRL) or red light pulses are hardly effective from 60 h after sowing onwards. Between 36 and 60 h after sowing the growth rate responds to red light pulses the effect of which is fully reversible by long wavelength far-red light. When seedlings are kept in cBL for 3 days and then treated with red light hypocotyl growth rate responds strongly. However, RL effectiveness decreases with time after transfer from BL to RL. BL → darkness transfer experiments with different levels of P_fr established at the beginning of darkness show that after a BL pretreatment phytochrome (P_fr) alone is capable of fully controlling growth rate. When white light (WL) is given no BL effect is detectable in weak WL. Only high light fluxes maintain a typical BL growth rate. At medium WL fluxes elongation rate returns gradually to the dark rate. The simplest explanation of the data is that light absorbed by a separate BL photoreceptor is necessary to maintain responsivity to P_fr. With increasing age of the seedlings the requirement for BL increases strongly. On the other hand, brief light pulses—given to demonstrate photoreversibility of phytochrome—remain equally effective provided that responsivity to P_fr exists.     

A POSSIBLE CONTROL BY A PHYTOCHROME-LIKE PHOTORECEPTOR OF CHLOROPHYLL SYNTHESIS IN THE GREEN ALGA Ulva rigida

Chlorophyll synthesis is stimulated by red light pulses in the green alga Ulva rigida C. Aghard. Chlorophyll synthesis in darkness is greater after longer red light pulses (30 min) than after shorter red light pulses (5 min). Chlorophyll synthesis was higher after red light pulses of 14 Wm_-2 fluence rate than after those of 7 Wm_-2. The effect of red light showed some far-red reversibility. The reversion by far-red light was higher after red light pulses of 4 min than after those of 30 min. These results indicate the existence of a rapid induction of chlorophyll synthesis during the red light pulses and a fast escape from photoreversibility. The percentage of reversion is also affected by the fluence rate of the light pulses. The reversion was reduced by about 15% when the photon fluence rate was increased from 7 to 14 Wm_-2. Reversion was also observed when red and far-red light pulses were applied successively. Thus, phytochrome or a phytochrome-like photoreceptor could be involved in the induction of chlorophyll synthesis in Ulva rigida.