INTERMEDIATES IN THE PHOTOCONVERSION OF FUNCTIONAL PHYTOCHROME IN FERN SPORES OF Dryopteris-I DEMONSTRATION AND QUANTITATIVE CHARACTERIZATION OF THE PHOTOCHROMIC SYSTEM Pr⇔ Ii700USING NANOSECOND-LASER PULSES*,†

Abstract— Spores of Dryopteris paleacea and D. filix-mas are positively photoblastic with an optimum in the action spectrum around 665 nm. Light is perceived by phytochrome and the relationship between germination and mole fraction of the far-red-absorbing form of this pigment, P_fr, was investigated with saturating irradiations between 662 and 747 nm under low-fluence-rate conditions. These control irradiations establish a proportion of the total phytochrome, P,_tot, as P_fr with P_fr/P_tot–φ at equilibrium. These φ -values were calculated according to data for native oat phytochrome (Kelly and Lagarias, 1985, Biochemistry 24, 6003) and the spectral characteristics of the interference filters. With this method a linear relationship could be found between φ and germination from 2 to 70% for D. paleacea and from 2 to 90% for D. filix-mas, if probit germination was plotted vs probit φ This correlation formed the basis of investigating the phytochrome photoconversion by dye-laser pulses of 380 ± 30 ns under high-fluence-rate conditions, and thus to test quantitatively the impact of the photoreversibility of intermediate reactions of the photoconversion and the red-absorbing form of phytochrome, P_fr on the final P_fr-level. Spore germination was initiated by a single-laser pulse in the range from 592 to 700 nm. The most effective wavelengths were 649 and 660 nm in both species, and at saturation maximal germination (ca. 50%) was obtained from 592 to 665 nm for D. paleacea or ca. 60% germination from 592 to 670 nm for D. filix-mas. Both saturation levels correspond to a ø-value between 0.40 and 0.45. This significantly diminished photoconversion is a consequence of the high-fluence-rate conditions during the laser pulse which establishes the photochromic system between P_r and a set of very early intermediates, Iⁱ_700, (= P_r? Iⁱ₇₀₀). This system can be described by the extinction coefficients of P_r and the intermediates Iⁱ₇₀₀, and by the quantum yields, 4,φ for the forward and reverse reactions as φ If φ is calculated, assuming a quantum yield of 1:1 for both reactions and with the extinction coefficients of P_r and Iⁱ_7(l() (= lumi-R) given by Eilfeld and Riidiger (1985, Z. Naturforsch. 40c , 109), significantly higher values are calculated for / as compared to φ found in the control experiments. These results can be explained either: (i) with a quantum yield ratio φ_pr-φ1700: φ_1700φpr=1:1 and an assumed additional dark reaction leading from Iⁱ₇₀₀ or later intermediates back to P_r: or (ii) with a quantum yield ratio φ_{pr φ 1700}: φ_{1700 φpr}=1:2. In this case all Iⁱ₇₀₀ have to relax to P_fr. In this case all Iⁱ₇₀₀ have to relax to P_fr.     

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.     

INTERMEDIATES IN THE PHOTOCONVERSION OF FUNCTIONAL PHYTOCHROME IN FERN SPORES OF Dryopteris–II. In vivo KINETICS OF THE DECAY OF Ii700 AND OF THE FORMATION OF Pfr STUDIED WITH A DOUBLE-LASER APPARATUS*

Abstract— Photoconversion of the red-light-absorbing form of phytochrome, P_r, to the far-red-light-absorbing form, P_fr, was investigated in vivo at 22°C with 600 or 800 ns laser pulses of high spectral purity and induction of spore germination in Dryopteris paleacea was used as indicator for the progress of photoconversion. This reaction is initiated by a saturating R-laser pulse of 648.5 nm, establishing an equilibrium of the photochromic system between P_r and the very early intermediates, Iⁱ₇₀₀ (P_rφ Iⁱ₇₀₀)- The decay of Iⁱ₇₀₀ as well as the formation of P_fr was recorded by the application of a second pulse varied between 698 and 717.5 nm, which inhibits the formation of P_lr being absorbed predominantly by Iⁱ₇₀₀or P_fr, respectively. The most effective inhibition for the second pulse is found up to 10 u.s after the first pulse and this is interpreted by photoreversion of Iⁱ₇₀₀ to P_r; thus reducing the formation of P_fr from Iⁱ₇₀₀. This early inhibition decreases between 10 μs to 100 ms after the R-laser pulse, as a result of the decay of Iⁱ_bl to a bleached species I,;. This decay can be described by three first order kinetics with the rate constants k₁₂= 16830 ± 2970 s^-1, k₁₂= 666 ± 218 s^-1,k₁₃= 9.8 ± 0.9 s^-1. A second inhibition, due to the formation of P_fr, is found for dark intervals <100 ms and can be described by two first order kinetics with the rate constants k₂₁= 2.9 ± 0.6 s^-1 and k₂₂= 0.17 s^-l.     

Primary Quantum Yield and Volume Change of Phytochrome-A Phototransformation Determined by Laser-Induced Optoacoustic Spectroscopy*

The primary quantum yield, Φ₁₇₀₀, for the photoconver-sion of the red-absorbing form of phytochrome, P_r, to the set of primary intermediates, Iⁱ₇₀₀, was redetermined by laser-induced optoacoustic spectroscopy at very low excitation laser fluences. The Iⁱ₇₀₀ value obtained is in the range of Φ_Pfr reported for the complete phototransformation P_r→ P_fr (J. M. Kelly and C. Lagarias, 1985, Biochemistry 24 , 4003–6010). An energy level of ca 150 kJ/ mol was found for the intermediates Iⁱ₇₀₀, i.e. ca 85% of the 0–0 level of P_r. Furthermore, a molecular expansion of 7 mL/mol (equivalent to 11 ų/molecule) was determined for the P_r→ Iⁱ₇₀₀ transformation. It reflects the protein reorganization induced by the geometrical pho-toisomerization of the chromophore, which results in changes of bonding interactions, in particular between the chromophore and its protein surrounding.     

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.     

THE KINETICS OF THE EARLY STAGES OF THE PHYTOCHROME PHOTOTRANSFORMATION Pr→ Pfr. A COMPARATIVE STUDY OF SMALL (60 kDalton) and NATIVE (124 kDalton) PHYTOCHROMES FROM OAT

A comparative study of the decay kinetics of photogenerated transients from small (60 kDalton) and native (124 kDalton) oat phytochrome in the red-absorbing form (P_r) in phosphate buffer containing 5 mM ethylenediamine tetraacetic acid, pH 7.8, (PB) and in PB containing 20% ethylene glycol, has been carried out in the temperature range 275–298 K. The analysis confirmed that at least two primary photoproducts, intermediates Iⁱ₇₀₀s and Iⁱ_7oo are formed from P_r. The kinetic parameters, as observed in PB at 695 nm and 275 K, are similar for the I₇₀₀ intermediates of both small and native phytochrome. Namely, the lifetimes are about 21 μs (component percentages 38%) for the I Iⁱ₇₀₀s and about 200 μ.s (62%) for the Iⁱ₇₀₀S- Arrhenius preexponential factors (A) of about 10¹⁶ and 10¹⁵ s-¹and activation energies of about 61 and 56 kJ/mol were measured for the absorbance decays of the I₇₀₀S of small and native phytochrome, respectively. The kinetic data favour parallel paths for the formation of the Iⁱ₇₀₀s from P_r, and the activation parameters indicate that the primary photoreactions of the transformation from P_r to the far-red-absorbing form are restricted to the chromophore within the protein. Moreover, the relatively modest temperature dependence of the lifetimes of the Iⁱ₇₀₀^S from small and native P_r supports the working hypothesis that the ground state reactions to the Iⁱ_bl, intermediates–although somewhat influenced by the polypeptide fragment that is removed upon degradation of native to small P_r–are localized to the chromophore, as is most probably the case also for the primary photoreactions. The effect of the addition of 20% ethylene glycol on the pre-exponential factors of the time-dependent decay functions is discussed in similar terms of the early stages of the phototransformation.     

STUDIES ON PHYTOCHROME PHOTOCONVERSIONS IN VITRO WITH LASER-INDUCED OPTOACOUSTIC SPECTROSCOPY*

After excitation at room temperature with a 15 ns dye laser pulse, phytochrome (60 kDalton) from etiolated oat seedlings was studied by optoacoustic spectroscopy, which records the heat emission caused by non-radiative deactivation processes of the photoexcited molecule. The action spectrum for the heat emitted by P_r deviates from the absorption spectrum around 610 and 695 nm, indicating the build-up of photoproduct(s) within 15 ns after excitation. It is proposed that the 695 nm product is identical with I₇₀₀ and lumi-R known to be the first intermediate on the P_r× P_fr pathway, and that the photochemical back reaction of I₇₀₀ to P_r occurs on the same time scale. About 90% of the absorbed light energy was lost by radiationless deactivation. Values for the rate constants of excited state deactivation of P_r and for the internal energy content difference of the primary photoproducts are calculated.     

FLUENCE RATE COMPENSATION OF THE PERCEPTION OF RED: FAR-RED RATIO BY PHYTOCHROME IN LIGHT-GROWN SEEDLINGS*

Abstract— The hypothesis that phytochrome functions as a sensor of vegetational shade through the perception of the red: far-red photon fluence rate ratio requires that the mechanism of perception be compensated for wavelength-independent fluctuations in fluence rate (Smith, 1982). This paper seeks to establish the lower limit of fluence-rate compensation and to assess whether or not compensation is effective at the total fluence rates typical of herbaceous canopies. Using specially-designed cabinets, Sinapis alba L. (white mustard) seedlings were grown from germination under a range of total photosynthetically-active radiation (PAR = 400 to 700 nm) values and a range of red: far-red ratios. The data indicate that fluence-rate compensation is effective above a PAR value of ca. 60 μ.mol m² s'. Pretreating seedlings at high red: far-red ratio and a PAR level of 300 (μmol m₂S_-1for increasing periods of time led to an extension of fluence rate compensation to lower fluence rates. The results are discussed in relation to the photosynthetic competence of the seedlings grown under these conditions.     

PROBIT ANALYSIS OF LOW AND VERY-LOW FLUENCE-RESPONSES OF PHYTOCHROME-CONTROLLED Kalanchoe blossfeldiana SEED GERMINATION*

Abstract— Kalanchoë blossfeldiana seeds are light-requiring for seed germination. On water or KNO₃ solution and irradiated with several daily red (R) irradiations, the seeds show a low-fluence (LF) response which is far-red (FR) reversible. Incubated on gibberellic acid (GA₃) the seeds show a very-low-fluence (VLF) response which can be saturated with red as well as with far red light. As germination is a quantal response, the sub-optimal segments of the dose-response curves are analysed by means of probit analysis in order to calculate the seed population parameters. There is a linear relation between the probit of the germination response and the logarithm of the fluence. Moreover, the slope for the VLF as well as for the LF response is the same. The VLF requires about 8 × 10⁴ times less fluence than the LF. VLF saturation with FR requires about 200 times more fluence than with R. Although, GA₃ and KNO₃ modulate VLF and LF, respectively, there is no direct influence on the phytochrome-phototransformations. Once P_fr is formed (in VLF or LF, or preserved in dry seeds) germination is proportional to the GA_S concentration (for VLF and dark germination) or proportional to the KNO, concentration (for LF). The non-photochemical events leading to germination seem to be triggered by a similar action mechanism for both GA, and KNO₃.     

Results of studies of potential light spectrum effects on human performance

Abstract— Sensitization of the phytochrome-mediated germination at 20°C of lettuce seeds (Lactuca sativa L. cv. Grand Rapids) by pretreatment at 4°C, 28°C, or on 1% ethanol, was studied. The 660 nm fiuence-response characteristics were similarly biphasic for all sensitizing treatments and displayed responses at very low fluences (VLFR) as well as responses characteristic of non-sensitized seeds at 10000-fold higher, low fluences (LFR). Maximum VLFR increased with the duration of sensitizing treatments. However, the fluence ranges required for the two types of responses remained relatively constant. These and additonal responses of sensitized seeds to 730 nm fluences were compared to simulations of a mechanism involving a receptor, X, and based on the dimeric structure of phytochrome in which each monomer is independently phototransformed from the inactive (P_r) to the active (P_fr) form. The fluence requirements for phytochrome photoconversion in seeds were determined to be similar to those of purified Avena phytochrome in vitro, on which photochemical parameters for the simulations were based. The analyses suggest that P_r:P_fr-Xand P_fr:P_fr-X are responsible, respectively, for the VLFR and the LFR, and that sensitization involves membrane influences on the activity of P_r:P_r-X. They also suggest the concentration of X to be about 0.001 that of total phytochrome dimer in this system.     

Photochemical inactivation of trypsin

Abstract —The quantum yield for inactivation of aqueous trypsin fits the expression φ_f=Σ_rf_rφ‘_r, where f_r, is the fraction of incident light absorbed by residues of type r and the φ’_r are constants. The values φ‘_trp= 0.012, φ_tyr= 0.005 and φ’_eys= 0.10, obtained at pH 3 in the wavelength range 240–290 nm, are attributed to independent events by comparing with quantum yields of the initial photochemical products and permanent residue destruction. The proposed inactivating processes are photoionization of one essential tryptophyl residue, photolysis of one essential cystyl residue, and splitting of an essential cystyl residue induced by light absorption in a nearby tyrosyl residue. The initial photochemical process from pH 3–7 identified by flash photolysis is the ejection of electrons from approximately two tryptophyl residues, leading to the formation of the disulfide bridge electron adduct and the hydrated electron. It is proposed that one photoionized tryptophyl residue is permanently disrupted and the other is restored through a back reaction that leads to a damaged, active enzyme form. An enhanced inactivation quantum yield at flash photolysis light intensities is attributed to a biphotonic process. A model based on one-photon photoionization of tryptophan from a short-lived precursor of the fluorescent state and the biphotonic photoionization of tryptophan via the triplet state is consistent with the experimental results.     

A flash photolysis–resonance fluorescence kinetics study of ground-state sulfur atoms: I. Absolute rate parameters for reaction of S(3P) with O2(3Σ).

The flash photolysis–resonance fluorescence technique has been used to measure the reaction of ground-state sulfur atoms with molecular oxygen as a function of both temperature and total pressure. The most suitable source of S(³P) for this study was found to be COS in the presence of CO₂, as a diluent gas and with the photolysis flash filtered so as to remove all radiation of wavelengths below 1650 Å. Under these conditions, it was found that over the temperature range of 252–423°K the rate data could be fit to a simple Arrhenius-type equation of the form Units are cm³ molec^?1 s^?1. The small A-factor for this reaction, the lack of any pressure dependence, and the direct observation of the production of O(³P) with increasing reaction time suggest that the S(³P) atom attacks the O₂(³Σ) molecule end-on forming SOO which rapidly falls apart to form SO (³Σ) and O(³P).     

Optical-limiting property of cyclic phenylacetylene oligomer synthesized under high pressure

The optical-limiting property of a new cyclic phenylacetylene oligomer was investigated. Toluene solutions of the oligomer contained within a quartz cell were irradiated with the pulse from frequency doubled Nd: Yag laser at 532 nm. At low input fluence, the transmittance of the toluene solution was constant and agreed well with that obtained by spectrophotometer. At high input fluence, the transmittance of the solution above a concentration of 5 wt % decreased with input fluence. The oligomer had an optical limiting property. As the concentration of the oligomer increased from 5 to 20 wt %, the threshold for optical limiting decreased. The threshold was independent of the molecular weight. The optical limiting property was analyzed by the following equation obtained according to the reverse saturable absorption mechanism; log(I₀/I) = K(I₀ - I) + A_g, where I₀ is the input fluence, I is the output fluence, K is a parameter depending on the absorption cross section and the relaxation time, and A_g is the absorbance of the ground state. © 1996 John Wiley & Sons, Inc.     

PHYTOCHROME AND EFFECTS OF SHADING ON GROWTH OF WOODLAND PLANTS

qrowth of Circaea lutetiaim plants was studied in various locations in or near a mixed deciduous woodland. Morphological changes resulting from increased shading included increases in leaf area ratio, specific leaf area and specific water content. Parallel measurements with a spectroradi-ometer confirmed that shading involved a reduction in both light fluence rate and light quality (e.g. red/far-red ratio). Phytochrome P_fr/P status was also studied by spectrophotometric measurements on Avena seedling test material and by biological (Lactuca seed germination) assay. Attempts were made to demonstrate phytochrome controlled changes in plant morphology under controlled environment, using both end-of-day far-red treatment and far-red enrichment of the main light period. Effects of natural shading were most clearly simulated by varying light fluence rate while maintaining a constant but high red/far-red ratio     

Yield and enrichment studies of C-13 isotope by multi-photon dissociation of Freon-22 at low temperatures

Multi-photon dissociation of Freon-22 (CF₂HC1) at low temperatures has been carried out to separate the C-13 isotope using a TEA CO₂ laser. Yield and enrichment of C-13 isotope in the product C₂F₄ are studied at 9P(22) laser line as a function of temperature (−50°C to 30°C). It is observed that at a given fluence when the temperature is lowered the yield decreases and the enrichment factor of C-13 increases. Room temperature irradiation of CF₂HC1 towards the blue edge of C-13 absorption (i.e. at 9P(20) laser line) gives low yield of the product (C₂F₄) at a fluence, which produces the desired enrichment factor of 100. An increase in fluence gives very high yield of C₂F₄ but the enrichment factor is very low. Irradiating CF₂HC1 at a temperature of −10°C enhances the enrichment factor to 100 and the yield obtained is comparable to that towards the red edge of C-13 absorption (i.e. at 9P(26) laser line). At a given enrichment factor higher enrichment efficiency is achieved when CF₂HC1 is irradiated at lower temperature.     

Chiral,Thermally Irreversible and Quasi-Stealth Photochromic Dopant to Control Selective Reflection Wavelength of Cholesteric Liquid Crystal

A chiral and thermally irreversible photochromic fulgide derivative incorporating an (R)-binaphthol unit in its acid anhydride moiety was used for the photoswitching of the pitch length of cholesteric liquid crystals. Since the absorption maximum wavelengths of both thermally stable photoisomers are nearly in the UV region (quasi-stealth photochromism), it can be exposed to visible light without inducing photochromic reactions. Therefore, when the photoswitching molecule is added to a permanent cholesteric liquid crystal whose reflection light wavelength is in the visible region, the UV light-induced photochromic reaction of the photoswitching molecule changes the wavelength of the reflection light in the visible light region. We have succeeded in regulating the color of cholesteric liquid crystalline cells between red and blue upon UV light irradiation. Attempts to introduce this system in polymer dispersed cholesteric liquid crystals are also described.     

A SHORT-LIVED INTERMEDIATE IN THE PHOTO-ENZYMATIC REDUCTION OF PROTOCHLOROPHYLL(IDE) INTO CHLOROPHYLL(IDE) AT A PHYSIOLOGICAL TEMPERATURE

The reduction of protochlorophyll(ide) into chlorophyll(ide) has been studied by flash absorption spectroscopy at 21°C, with a time resolution of 0.5 µs. The absorption changes have been recorded in the range 670–720 nm after the first and subsequent flashes given to an extract of etiolated bean leaves. At 695 nm the flash-induced absorption increase has its maximum value immediately after the flash and then partly decays with a half-time of about 7–10 µs. A complementary behaviour is observed at 675 nm where the absorption change is very small at time zero and then increases to a stationary value with a half-time of about 6–9 µs. From measurements at several wavelengths it is concluded that a species with an absorption peak around 695 nm is formed immediately after the flash and is then transformed into a stable species with an absorption peak around 675 nm. Measurements at lower temperatures, down to—50°C, show that the transformation is much slowed down at decreased temperatures. The species absorbing at 695 nm (P₆₉₅) is attributed to an intermediate in the photoreduction of protochlorophyll(ide) P_639,650 into chlorophyll(ide) P₆₇₆. When the protochlorophyll(ide) is photoreduced before the flash illumination, the newly formed chlorophyll(ide) gets to a triplet state, which decays with a half-time of 15 µs at 21°C. This result shows that carotenoid molecules do not exert their protective role at this stage of chlorophyll (Chi) formation.     

CO2 laser excitation of triethylsilane: Time resolved luminescence of diethylsilyl radical

The infrared multiphoton excitation of triethylsilane in the gas phase, with a pulsed CO₂ laser at high intensities (I > 700 MW/cm²), produced an intense luminescence. The spectrum and time profile of this luminescence was studied as a function of pressure, and laser frequency. The radiative lifetime of this emission was 357 ± 10 ns, and the quenching rates by Cl₂ and NO were determined from lifetime measurements. A reasonable mechanism for the interpretation of this luminescence involves the initial infrared multiphoton decomposition of triethylsilane, followed by the secondary infrared multiphoton excitation of the primary photofragment diethylsilyl radical, which subsequently undergoes relaxation to an excited electronic state. The addition of O₂ resulted in a new chemiluminescence at shorter wavelengths, which corresponds to the SiO* chromophore group. © 1994 John Wiley & Sons, Inc.     

KINETICS OF Pfr APPEARANCE IN Amaranthus caudatus‡

Abstract— The kinetics of the far-red absorbing form of phytochrome (P_fr) appearance from intermediates in the pathway from the red absorbing form of phytochrome (P_r) to P_fr that accumulate under high fluence rate white light have been investigated in 3-day old dark grown Amaranthus caudatus seedlings. The appearance of P_(r after a 5 s white light pulse was measured over the temperature range -8 to 25^°C in samples flushed with O₂ or N₂. Over the whole temperature range under anaerobic conditions the kinetics of the slowest component of P_fr appearance are faster than in the presence of O₂. Arrhenius plots are linear over this temperature range and indicate the activation energy for the slowest component of P_fr appearance is 44.05 ± 1.97 kJ mol^?1 for O₂ and 53.69 ± 4.86 kJ mol^?1 for N₂.     

The quenching of excited iodine atoms by oxygen molecules as studied by opto-acoustic spectroscopy

The opto-acoustic spectrum of I₂ in the presence of various quenching gases — NO, O₂, CH₃I, SO₂, C₃H_S, N₂, and He — has been studied. Of these, the I₂/O₂ spectrum is quite different due to the near-resonant energy transfer I(²P$\text{1}\text{2}$) + O₂(³Σ) → I(²P$\text{3}\text{2}$) + O₂(^IΔ), wherein the resistance of the O₂((^IΔ) species to collisional relaxation severely distorts the acoustic signal. The photochemical production of excited ²P$\text{1}\text{2}$ iodine atoms commences at wavelengths considerably longer than the dissociation limit of the I₂B? state.