IN VIVO and POST MORTEM MEASUREMENTS OF THE ATTENUATION SPECTRA OF LIGHT IN MAMMALIAN TISSUES

Abstract— The attenuation of light in rabbit liver and striated muscle and in pig brain has been measured in the wavelength range400–800 nrn. A variable-wavelength monochromatic light source was used to illuminate the surface of the exposed tissues in situ. Optical fibers, coupled to a single photon counter, were positioned within the tissues at different depths to determine the distribution of light flux along the central axis of the light beam. The wavelength-dependence of attenuation was measured in vivo, and changes occurring in the attenuation spectra immediately post mortem were recorded. Strong absorption bands were observed around 425 nm and 550 nm in all three tissues, both in vivo and post mortem. At longer wavelengths, the attenuation decreased slowly, and no significant structure was observed. Substantial changes occurred immediately post mortem, particularly in the absorption bands. These effects are attributed mainly to de-oxygenation of blood, and changes in blood concentration in the tissues. The implications for light dosimetry in photodynamic therapy are discussed.     

THE EFFECT OF RED AND FAR RED LIGHT ON CAROTENOID AND CHLOROPHYLL FORMATION IN PEA SEEDLINGS

Abstract— Treatment of etiolated pea seedlings with a short exposure to red light caused a stimulation of growth (size and dry wt production) and carotenoid synthesis during the following 48 hr compared with seedlings kept entirely in darkness.The effect is nullified by a following dose of far red light and thus the phenomenon is probably phytochrome-controlled.
Similar treatment with red light one hour before continuous illumination with white light tended to reduce the lag period for chlorophyll synthesis.Again a following dose of far red light reversed this response.     

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.     

PICOSECOND DYNAMICS OF THE EXCITED STATE RELAXATIONS IN PHYTOCHROME

Abstract— A comparative study of the picosecond kinetics of rye ( Secale cereale L.) phytochrome, its 39 and 23 kDa chromopeptides and deuterated rye phytochrome has been carried out. Evidence is presented that the fluorescence decay of Pr contains a very short lifetime component (14 ps) which has escaped detection in the fluorescence studies reported so far. Thus, the overall decay is well described by four exponential components, two rapid (14 and 44 ps) and two slower ones (157 and 690 ps). The fluorescence decays of deuterated Pr and of a 39 and 23 kDa chromopeptide of Pr also require the analysis in terms of four exponentials for a good fit. Some of the lifetime and amplitude values obtained differ significantly from the values estimated for Pr. In the chromopeptides, the two longer components have distinctly slower decays. For the two faster components the lifetimes remain approximately the same, but their relative amplitudes vary greatly. In deuterated Pr, the lifetimes are affected only slightly by deuteration. In contrast, the decay amplitudes are strikingly altered. Moreover, from a rate equation simulation modelling the observed fluorescence kinetics, it turns out that the yields for the various deactivation steps in the chromopeptides and in deuterated Pr reveal differences from the corresponding values in Pr. The implications of the results presented with respect to the influence of the protein moiety of Pr on the picosecond relaxation process are discussed.     

THE IN VIVO SPECTROPHOTOMETRIC ASSAY OF PHYTOCHROME IN TWO MATURE DICOTYLEDONOUS PLANTS*

Abstract— Mature Sinapis alba L. and Impatiens parviflora DC. were treated with the herbicide norfiuorazon prior to development of the third or second leaf, respectively. This treatment yielded a partially bleached plant capable of normal growth and development. The bleached leaves were used for spectrophotometric phytochrome assay. In mature plants an almost constant level of phytochrome is maintained under continuous white light. The dark kinetics and the response of the phytochrome system to light of various qualities provide further evidence of the stable character of the phytochrome system.     

PHOTOXIDATION OF ASCORBATE TO OXALATE AND THREONATE MEDIATED BY CHLOROPLASTS IN FAR RED LIGHT

Abstract— Chloroplasts illuminated with light absorbed predominantly by Photosystem I can mediate the photoxidation of ascorbic acid to threonic and oxalic acids. Two micromoles of oxygen are consumed for each micromole of ascorbic acid added. The kinetics of this endogenous chloroplast system closely resemble the kinetics of an artificial flavin-manganese-catalase system in which flavin is the photosensitizing dye.     

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.     

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.     

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.     

THE EFFECT OF PROLONGED LIGHT EXPOSURE ON THE EFFECTIVENESS OF PHYTOCHROME IN ANTHOCYANIN SYNTHESIS IN TOMATO SEEDLINGS*

Abstract— The hypocotyl of the tomato ( Lycopersicon esculentum ) seedling synthesizes large amounts of anthocyanin if exposed to prolonged light. Single light pulses are totally ineffective. The involvement of phytochrome can be shown by light pulse treatments following a prolonged light exposure. It is predominantly the action of blue/UV light which leads to a high responsiveness of anthocyanin synthesis towards phytochrome. Moreover, the data suggest a phytochrome-independent action of blue/UV light, in particular of UV-B, on anthocyanin synthesis.     

THE FUNCTION OF PHYTOCHROME IN THE NATURAL ENVIRONMENT—IV. LIGHT QUALITY AND PLANT DEVELOPMENT

Abstract— Studies of the effect of light quality on the growth of Cucurbita pepo L . and Chenopodium album L . showed that large changes in morphological development can be induced by altering the proportion of red and far-red in the incident radiation. Although phytochrome photoequilibria established at the beginning of the dark period affected development, similar changes were achieved with long term and continuous irradiation. The change in the growth pattern of C. album (a common weed species in cultivated land) caused by simulated wheat canopy shade, appeared to involve a redirection of growth towards processes which would achieve greater height at the expense of lateral growth. The evidence presented supports the hypothesis that phytochrome may be involved in the detection of mutual shading and the modification of development in an appropriate manner.     

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.     

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.     

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.     

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.     

INTERACTION BETWEEN PHYTOCHROME AND THE BLUE LIGHT PHOTORECEPTOR SYSTEM IN Mougeotia*

Abstract— Face-to-profile chloroplast movement in Mougeotia was induced by sequences of strong blue and red short irradiations. This type of response occured only when blue light was applied prior to or simultaneously with red light, and far-red irradiation was necessary after the sequence to cancel the remaining gradient of the far-red absorbing form of phytochrome P_fr. The dependence of the response magnitude on blue and red light sequences was studied for a wide range of light durations and dark intervals. The relationship between the response and the dark interval points to the lack of direct coupling between phytochrome and blue-absorbing “cryptochrome”. It was postulated that a photoproduct having a life-time of2–3 min is formed by the blue-light-mediated reaction. This photoproduct interacts with phytochrome during its transformation or with its final P_fr form.     

THE ENHANCEMENT OF PHYTOCHROME ACTION ON BARLEY LEAF UNFOLDING BY INTERMITTENT LIGHT

Abstract. The phytochrome controlled unfolding of etiolated barley leaves proceeds best if orange light (0.6 J/m²/s; 625 ± 25 nm) is given as two appropriately timed short pulses rather than as a single longer light period.     

THE MODE OF INTERACTION BETWEEN BLUE (UV) LIGHT PHOTORECEPTOR AND PHYTOCHROME IN ANTHOCYANIN FORMATION OF THE SORGHUM SEEDLING

Two non-photosynthetic photoreceptors (phytochrome and a blue light photoreceptor) are involved in light-mediated anthocyanin synthesis in the mesocotyl of Sorghum seedlings. The present study was undertaken to investigate the kind of interaction between phytochrome and the blue light photoreceptor. The data show that phytochrome (P_fr) can only act once a blue light effect has occurred. On the other hand, the blue light effect cannot express itself without P_fr. It is concluded that there is an obligatory dependency (or sequential interaction) between the blue light effect and the light effect occurring through phytochrome, although the blue light photoreaction per se is not affected by the presence or absence of phytochrome. The latter statement is based on the results of dichromatic experiments, i.e. simultaneous, high fluence rate irradiation with two kinds of light. Blue light can be replaced by UV light. It is not clarified yet whether the effect of blue and UV light is due to the same photoreceptor.