PHYTOCHROME INDUCTION OF PHOTOREACTIVATING ENZYME IN Phaseolus vulgaris L. SEEDLINGS*

Abstract— Photoreactivating enzyme (PRE) activity was measured in hypocotyls of Phaseolus vulgaris L. seedlings using a radioimmunoassay for thymine dimers. In dark-grown seedlings a five-fold increase in PRE activity was observed after 6 h of irradiation with blue or far-red light. Short time irradiations with red light were also effective. Reversibility of this red-light-effect by a subsequent short term irradiation with far-red light and also the high effectiveness of continuous far-red light indicate that PRE activity is under phytochrome control. This observation points to PRE induction via gene activation.     

CONTROL BY PHYTOCHROME OF CHLOROPHYLL SYNTHESIS IN SEEDLINGS OF SORGHUM VULGARE

The strong effect of light pretreatments on the synthesis of chlorophyll-a and-b in the shoot of Sorghum vulgare (kept under saturating white light) can be attributed to phytochrome only. No specific blue light effect was found. The phytochrome system appears to function perfectly normally under these conditions. Escape from reversibility is not detectable up to approximately 40 min after the onset of an inductive red light pulse. Thereafter, escape is fast, being completed at approximately 2.5 h after the inductive light pulse.     

PHYTOCHROME CONVERSION AS AN IN SITU ASSAY FOR EFFECTIVE LIGHT GRADIENTS IN ETIOLATED SEEDLINGS OF Zea mays

Abstract— In situ photochemical conversion and measurement of phytochrome can be used as a non-intrusive assay for axial light conduction along etiolated plant tissues of Zea maysL. A computerized single beam spectrophotometer was used to measure phytochrome conversion just below the coleoptilar node following irradiation of the plant shoot with 632 nm light at various distances from the measurement point. Photochemical conversion decreased log-linearly with increasing distance between the irradiation and measurement points. The distance over which the light was attenuated by 50% was 1.80 mm for mesocotyl, 1.60 mm for coleoptile plus primary leaf, and 1.15 mm for primary leaf alone (coleoptile removed). These results verify the photochemical significance of axially conducted light and thereby provide supporting evidence that such light conduction is important for photomorphogenesis.     

ON THE POLARITY OF PHYTOCHROME CONTROLLED LEAF UNFOLDING IN DARK-GROWN SEEDLINGS OF TRITICUM AESTIVUM*

Abstract— The apical ends of sections cut from etiolated primary wheat leaves show a higher degree of phytochrome-mediated leaf unfolding than the basal ends, regardless of the position of the segment in the leaf. It could be clarified that this polarity is not the result of a stable gradient within the intact dark grown leaf. Partial irradiation experiments have shown that leaf unfolding can be induced much more effectively by an irradiation of the apical half of a section than of the basal half. Though only the basal end of the sections was incubated with gibberellic acid (GA₃) in darkness, the degree of the GA₃-induced leaf unfolding was highest at the apical end of these non-irradiated segments. The results suggest that in each separate leaf section a new gradient of a still unknown “unfolding-factor” is established.     

PHYTOCHROME STATES IN ETIOLATED PEA SEEDLINGS: FLUORESCENCE AND PRIMARY PHOTOREACTIONS AT LOW TEMPERATURES

Emission spectra of the red phytochrome form (Pr) and fluence time-response curves of the Pr fluorescence intensity changes were measured in etiolated pea seedlings at low temperatures (80–150 K) in connection with its phototransformations into the initial photoproduct (Lr) and back upon actinic red (667 nm) and far-red (696 nm) illumination. The variable fluorescence reaches 45% at 85 K and decreases with the rise of temperature. Three kinetic components of the changes were found in the direct (Pr→Lr) and back (Lr→Pr) photoreactions belonging to three states of phytochrome: “slow”, “fast” and “very fast” (respective indices: s, f and vf). The amplitudes of the components and rate constants to reach photoequilibrium were determined in the direct and back photoreactions at different temperatures, and from this, their quantum yields, extent of the Pr?Lr phototransformation and activation energy of the reactions were evaluated for the three Pr and Lr states. The yields differ from each other by approximately a factor of 10 and those for the direct and back photoreactions are close to each other. The proportion of the amplitudes of the variable fluorescence of the three phytochrome states changes with temperature and upon the Pr→Lr photo-transformation and the Pr states differ in the position of their emission spectra by 3–5 nm. A close similarity between the Pr and Lr properties was observed, which implies a symmetrical scheme of their photoreactions. It is suggested that the three phytochrome species may originate in different conformational states of the chromophore and they independently transform in parallel photoreactions into the respective photoproducts: Prvf?Lrvf, Prf?Lrf and Prs?Lrs.     

CIRCANNUAL COURSE OF PHOTOMORPHOGENETIC REACTIVITY IN ETIOLATED BEAN SEEDLINGS

Hook opening and leaf expansion, measured 24 h after standard red light illumination, were considered as parameters determining the photoactivity of etiolated Phaseolus vulgaris L. seedlings. The experiment was repeated every h during one 17-h period each month for 1 year. The mean values for each experimental period indicated that the photoactivity of the etiolated seedlings changed markedly throughout the year according to a seasonal pattern.     

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.     

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.     

CHANGES IN THE RATES OF PHOTOCONVERSION OF PHYTOCHROME DURING ETIOLATION IN MUSTARD SEEDLINGS

Abstract— The relative phytochrome photoconversion rates in cotyledons and hypocotylar hook of etiolating mustard ( Sinapis alba L.) seedlings were measured between 16 and 96 h after sowing. It was found that at constant fluence rates photoconversion rate in red light increases in both organs with time whereas the photoconversion rate in far-red (756 nm) light decreases with time of development. Since the isosbestic point remains constant, it was concluded that the observed changes cannot be attributed to changes of extinction coefficients. It was not possible, however, to decide whether the observed changes are due to changes of light attenuation or quantum yields.     

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.     

IN VITRO SYNTHESIS OF PHYTOCHROME APOPROTEIN DIRECTED BY mRNA FROM LIGHT AND DARK GROWN AVENA SEEDLINGS*

Abstract— The aim of this work was to compare the translatability of poly-A-RNA from light- and dark-grown Arena seedlings, the product of which should be phytochrome. Polysomal poly-A-RNA was isolated from 4-day-old dark-grown Avena seedlings (Avena sativa L. cv. Garry) and translated in a rabbit reticulocyte lysate system. Immunoprecipitation by anti-phytochrome serum was used for measurements of specific phytochrome translation. Characterization of the translated and immunoprectpitated protein was performed by comparison with [₃₅S]-methionine in vivo-labelled phytochrome. The specificity of the precipitation was shown by parallel use of non-specific serum and by competitive inhibition of precipitation by exogenous unlabelled phytochrome isolated by affinity chromatography.     

CHLOROPHYLL METABOLISM IN ETIOLATED GHERKIN SEEDLINGS I. PHOTOINHIBITION OF CHLOROPHYLL ACCUMULATION

Abstract. Cotyledons of etiolated gherkin seedlings do not turn green upon transfer to high intensity red light (about 25 W/m²). A pre-irradiation with high intensity red light has an after-effect as chlorophyll accumulation during a subsequent exposure to white light (20 W/m²) is inhibited.
The capacity of protochlorophyll regeneration during a dark period depends on the length of a previous light period but is hardly affected by the light intensity. At high intensity light the rate of protochlorophyll regeneration, which also depends on the length of the foregoing irradiation, is lower than that at low intensity light only during the first 1.5h of the light period. It is concluded that high intensity red light inhibits chlorophyll accumulation mainly by photo-bleaching of chlorophyll. The after-effect is the result of a photooxidation which may lead to photo-bleaching of newly formed chlorophyll in relatively low intensity light.
Photoinhibition of chlorophyll accumulation is accompanied by a disturbed development of etioplasts into chloroplasts.     

PREVENTION BY PHYTOCHROME OF PHOTODELAY IN CHLOROPHYLL ACCUMULATION

A photodelay in chlorophyll- a and -b accumulation is observed in mustard ( Sinapis alba L.) cotyledons during the first 3 h after the onset of white light even at medium light fluxes (3500 lx). A pretreatment of two red light pulses or a 12 h far-red light pretreatment, both operating through phytochrome. prevent the photodelay completely. This is a specific phytochrome effect since'it can be separated from the effects of phytochrome on the rates during pre-steady state and steady state phases of chlorophyll accumulation in saturating white light. Thus, photostability of chlorophyll in nature is a photoresponse mediated through phytochrome.     

RELATIVE IMPORTANCE OF BLUE LIGHT AND LIGHT ABSORBED BY PHYTOCHROME IN GROWTH OF MUSTARD (Sinapis alba L.) SEEDLINGS*

Abstract— Hypocotyl straight growth in mustard (Sinapis alba L.) responds very strongly and in precisely the same way to low fluence rate red (RL) and white light (WL). The effect of weak light can be attributed fully to light absorption by phytochrome. Only with increasing fluence rate an effect of blue light (BL) comes into play which cannot be explained by the action of phytochrome. However, this specific action of BL can be demonstrated in hypocotyl growth of mustard seedlings only up to 5 days after sowing (25°C). With older seedlings control of hypocotyl growth seems to be exerted exclusively via phytochrome. Regarding the far-red light dependent “high irradiance reaction” (FR-HIR) it was found that it plays a dominant role in growth of mustard only during a relatively short period. It tends to disappear in favor of a RL-HIR between 3 and 4 days after sowing. It is concluded that the seedling exhibits a largely endogenous temporal pattern of responsiveness to light. Phototropism of the mustard seedling can be elicited by low fluence rates (< 1 mW m^?2) of unilateral BL. This same light has no effect on straight growth. It is concluded that BL-dependent phototropic growth response of a hypocotyl and the effect of BL on longitudinal growth of the hypocotyl are unrelated phenomena.     

PHOTOCONTROL OF PHYTOCHROME DESTRUCTION IN GRASS SEEDLINGS. THE INFLUENCE OF WAVELENGTH AND IRRADIANCE

Abstract— –The kinetics of phytochrome destruction in vivo of coleoptiles and mesocotyls of etiolated grass seedlings (Avena sativa L., Zea mays L.) in continuous light were investigated using wavelength and irradiance as experimental variables. In contrast to dicotyledonous seedlings, the destruction reaction of these monocotyledons is saturated at very low levels of the far-red absorbing form of phytochrome, P_fr (e.g. at 1% of total phytochrome, corresponding to the photostationary state established by 727 nm light, in 2.5-day-old dark-grown Avena). On the other hand, the first-order rate constant of monocotyledon destruction may be at least one order of magnitude larger than in dicots, as indicated by the zero-order rate measured in the presence of saturating amounts of P_fr (τ_l/2 1.5 min in Avena). At sub-saturation P_fr levels, the destruction rate was found to be determined by the rate constants of the photoreactions over a wide range of wavelengths and irradiances. These results can be interpreted in terms of a destruction enzyme with high catalytic efficiency but limited availability. Analysis of in vivo binding of phytochrome to a pelletable cell structure during destruction revealed that both the pelletable and the non-pelletable fraction lose photoreversiblility with similar rates and thus provide no useful information with respect to a causal relationship between the two processes. However, due to the short half-life of P_fr at sub-saturation levels (which make the photoreactions and intermediary processes rate-limiting for destruction even at relatively high irradiances) the existence of a similarly rapid dark-reaction between the photoreactions producing P_fr and the destruction reaction could be demonstrated. This dark reaction displays the properties of P_fr binding to a receptor site.     

TWO SPECTROSCOPICALLY AND PHOTOCHEMICALLY DISTINGUISHABLE PHYTOCHROMES IN ETIOLATED SEEDLINGS OF MONOCOTS AND DICOTS *

Abstract Fluorescence (F) emission spectra of the red-absorbing phytochrome form (Pr) at 85 K, temperature dependence of the F intensity and the extent of the Pr F changes in the phototransformation of Pr into the first stable photoproduct (lumi-R) at 85 K (γ₁,) and into the far-red-absorbing form (Pfr) at 267 K (γ₂) were investigated in etiolated shoots and roots of monocots (oat, maize, rice) and dicots (pea, cress). These characteristics monotonously changed as a function of the phytochrome content, [Ptot]: with its decrease to 3-5% of the maximal values, the F spectrum shifts from 686 nm to 682 nm, its half-band width rises from 22 nm to ca 25 nm, temperature dependence of Pr F changes its character, γ¹, drops down from ca > 0.45 to ca 0.05-0.10 and γ₂ from 0.80–0.82 to ≤0.70. These data were interpreted in terms of two different phytochromes whose relative concentration varies with [Ptot]: (1) a longer wavelength type with the F maximum at 686 nm, low activation energy of the photoreaction (Ea ≤ 3–4 kj/ mol) and high extent of the phototransformation at 85 K (0.49 ± 0.03) and at 267 K (ca 0.85) (Pra); (2) a shorter wavelength type practically inactive at 85 K with F maximum at 682 nm, higher Ea (ca 35 kj/mol) and lower extent of the Pr & Pfr phototransformation (≤0.70) (Pri). [Pra] widely varies in different parts of the seedlings (up to 100 times) and Pra dominates when [Ptot] is high. The [Pri] is much more constant (variations, <10 times), and it becomes the major one when [Ptot] drops down. The two species are likely to belong to the labile (type 1) and stable pools of pigment and not to be connected with the localization of the pigment in the cell since red-far-red preillu-mination, which is believed to bring about sequestering of the pigment, does not change their relative concentration and properties.     

REGULATION OF PROTEIN PHOSPHORYLATION BY PHYTOCHROME IN Sorghum bicolor

Abstract— In vitro phosphorylation of some polypeptides was affected in extracts obtained from 5-and 6-day-old plants irradiated with 5 min of red light. The phosphorylation of 55 kDa polypeptide in both 5- and 6-day-old plants, a 60 kDa, and 76 kDa polypeptide in 6-day-old plants and 70 kDa, 67 kDa polypeptide in 5-day-old plants was stimulated by red light. This effect was reversible by far-red light. The extent of stimulation by red light and reversal by far-red light varied for different polypeptides. No differential effect of red and far-red light was seen on the phosphorylation of 94 and 40 kDa polypeptides. In fact, phosphorylation of 94 kDa polypeptide in 6-day-old plants decreased on red light irradiation. These results show that the phosphorylation or dephosphorylation of some proteins is affected by phytochrome and the effect of light is also dependent on the age of the plant.     

INVOLVEMENT OF PHYTOCHROME IN LIGHT CONTROL OF STEM ELONGATION IN CUCUMBER (Cucumis sativus L.) SEEDLINGS

A series of polycondensed aromatic N-heterocycles (acridine, benzo-f-quinoline 1,2,7,8-dibenzacridine and 3,4,5,6-dibenzacridine) were adsorbed from the gas phase and from liquid solution on highly dispersed silica gels with very different specific surface areas and pore sizes. The translational mobility of the adsorbed species was quantified by the triplet decay and the delayed fluorescence following bimolecular triplet-triplet annihilation after pulsed laser irradiation. The decay kinetics were analyzed with conventional second order rate equations and with the fractal approach. The first method is reliable without limitations on adsorbents with large pore diameters. It yields second order annihilation constants of 4 times 10¹²-6 times 10¹¹ dm² mol^?1 s^?1 depending on the masses and sterical requirements of the adsorbates. For the second method a spectral dimension d_s= 4/3 was used. This method is quantitatively applicable to all heterocycles adsorbed on silica gel 60 that have very small pore sizes. An activation energy of 4.9 ± 0.5 kcal mol^?1 was obtained for the translational diffusion of acridine on hydroxylated silica gel.     

PHYTOCHROME DESTRUCTION IN DARK - AND LIGHT-GROWN AMARANTHUS CAUDATUS SEEDLINGS

Abstract— Slow destruction of the far-red-absorbing form of phytochrome (P_fr), which has been observed in light-grown oat and maize, occurs in light- and dark-grown Amaranthus, Pharbitis , and Brassica seedlings as well. Destruction of P_fr in these seedlings shows two phases: if a high level of P_fr is produced in dark-grown seedlings, the destruction is fast in the beginning and then slows after a low P_fr level has been reached. Slow P_fr destruction is predominant in light-grown tissue.     

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.