FLUORESCENCE OF PHYTOCHROME IN THE CELLS OF DARK-GROWN PLANTS AND ITS CONNECTION WITH THE PHOTOTRANSFORMATIONS OF THE PIGMENT

Abstract Fluorescence of phytochrome is found in the cells of etiolated monocotyledonous and dicotyledonous plants. The red light-absorbing form of phytochrome (P_r) fluoresces at 77 K with a yield 0.3±0.1 and maxima at 672–673 nm and 684–686 nm in the excitation and emission spectra, respectively. The emission is characterized by the sharp temperature dependence of its intensity, its high (～ 40%) polarization, and the violation of the mirror symmetry rule. Connection of the fluorescence with P_r photoreactions is followed in the interval 77–293 K. A P, photoproduct, lumi-R, is fluorescent with maxima at 696 nm and 705 nm in the excitation and emission spectra; the far-red light absorbing form of phytochrome (P_fr) is practically nonfluorescent. Three isochromic emitting P_r species are present differing in their photochemical properties: P_r1 and P_r2 which phototransform irreversibly and reversibly at T 170 K into lumi-R, and lumi-R₂, respectively, and P_r3 which undergoes photoconversion only at T > 240 K. The activation energies of P_r2 and P_r3 photoreactions are evaluated to be 2.9–3.3 kJ/mol and 26 kJ/mol. Complex dynamics of changes of P_r fluorescence and of the extent of its decrease in the photoconversion P_r? P_fr in germinating pea and bean seeds suggests the existence of two P_r pools one of which is incapable of P_r? P_fr phototransformation. Thus, the developed fluorescent method of phytochrome assay and investigation in the cell revealing multiplicity of phytochrome states in vivo proves to be very sensitive (about 1 ng) and informative.     

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.     

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.     

THE EFFECT OF DEUTERIUM OXIDE ON THE FLUORESCENCE AND PHOTOTRANSFORMATION OF 124 kDALTON PHYTOCHROME*

Abstract— To probe the nature of primary photoprocess and the mechanism of the phototransformation of undegraded 124 kDa oat phytochrome, solvent deuterium isotope effects on the fluorescence and phototransformation of phytochrome have been investigated. The fluorescence intensity and lifetime of phytochrome (P_r form) are greater in D₂0-buffer than in H₂O-buffer, suggesting a possible involvement of proton transfer in the primary photoprocess of phytochrome. Although the photostationary equilibrium (P_r to P_fr ratio) was not altered by deuterium oxide, in contrast to degraded phytochrome, the rate constants of both transformations, P_r→ P_fr and P_fr→ P_r were enhanced by up to 24%. The P_r to P_fr phototransformation of degraded phytochrome, however, was retarded by about the same percentage in D₂O. These opposite effects of D₂O with degraded and undegraded phytochromes underscore the fact that the P_r form from the former reverts to the P_r form in the dark, apparently catalyzed by deuterated general and/or conjugate acidic group(s). With the degraded phytochrome the deuterium oxide enhancement of the rate of dark reversion was approximately 2-fold (Sarkar and Song, 1981). Both the fluorescence intensity and the rates of phototransformation of phytochrome were enhanced in D₂O with successive photocyclings (P_r→ P_fr→ P_r→ P_fr→ P_r etc.) with alternating red and far-red irradiation. It has been proposed that successive photocycling of phytochrome in D₂O results in proton-deuteron exchange in the partially exposed P_tr chromophore and/or its surrounding amino acid residues.     

PHYTOCHROME PHOTOCONVERSION in vivo. EFFECT OF THE INITIAL Pfr/Ptot RATIO

The equation for phytochrome photoconversion, derived from photoconversion kinetics of purified phytochrome, predicts that the rates of photoconversions starting from low and high Pfr/Ptot₍₀₎, (initial Pfr/Ptot) should be the same for light of the same quality and fluence rate. The situation might be different in vivo. Phytochrome photoconversion rates were measured in excised cotyledons of Cucurbita pepo L. exposed to BL (blue; ?_BL= 0.39; ?, Pfr/Ptot at photoequilibrium) and RI (mixture of red and far red; ?_RI= 0.46) after saturating preirradiations with red and far-red to establish high (0.78) and low (0.02) Pfr/Ptot₍₀₎, respectively. Under BL, the rate of photoconversion is faster when starting from a high than a low Pfr/Ptot₍₀₎; under RI, the rate of photoconversion is faster when starting from a low than a high Pfr/Ptot₍₀₎., No effects of Pfr/Ptot₍₀₎, on photoconversion rates were found in phytochrome solutions exposed to BL and RI. These data provide another indication of the discrepancies between phytochrome photconversion kinetics in vivo and in vitro.     

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.     

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.     

Stabilization of phytochrome intermediates by low temperature

Abstract— The photocon versions between the red-absorbing form (P_r) and the far-red absorbing form (P_fr) of phytochrome were examined at low temperatures. Partially purified preparations of the chromoprotein were examined in phosphate buffer and in 25 per cent buffer plus 75 per cent glycerol. Actinic irradiation of P, below – 150°C produces an intermediate with maximum absorbance near 695 nm, R₆₉₅. Actinic irradiation of R₆₉₅ converts it back to P. Above – 150°C R₆₉₅ decays to a low extinction form of phytochrome, R, which in turn decays to P_fr upon further warming. Light absorption by P_fr below – 150°C results in the formation of an intermediate form of phytochrome with maximum absorbance near 660 nm, FR₆₆₀. FR₆₆₀ decays upon warming to a lower extinction form, FR'. which in turn decays to P_r on continued warming. No evidence was obtained to suggest that any of the observed intermediate states are involved in more than one direction of phytochrome photocon version.     

INDUCTION OF SEED GERMINATION IN Lactuca sativa L. BY NANOSECOND DYE LASER FLASHES*,†

Abstract— A 15 ns, tunable dye laser was used to induce germination of the photoblastic seeds of Lactuca sativa. One red laser flash in the range from 620 to 690 nm was sufficient to increase germination significantly above the dark level. Repeated flashes, however, were necessary to saturate the physiological response. The wavelength dependence for induction of germination differed for single and repetitive flashes. After saturating far-red irradiation, the effect of single-flash induction was a function primarily of the absorption spectrum of P_r. In addition, the establishment within the lifetime of a flash of a photochromic system between the red absorbing form of phytochrome (P_r) and the sum of photoreversible intermediate forms (ΣI₇₀₀) contributes to this wavelength dependence at high fluence rates. This photochromic system is assumed to be shifted significantly toward P_r by wavelengths 660 nm. Similarly, a strong double-flash effect, which is seen as an increase in effectiveness when a given total fluence is provided by two consecutive flashes rather than by one flash only, is restricted to those wavelengths that considerably shift the photochromic system P_r?ΣI₇₀₀ toward P_r. Finally, the saturation level produced by a series of laser flashes depends, additionally, on absorption by P_fr.     

Comparison of the protein conformations between different forms (Pr and Pfr) of native (124 kDa) and degraded (118/114 kDa) phytochromes from Avena sativa

Abstract— Circular dichroic properties of native, 124 kDa phytochrome from etiolated Avena sativa seedlings have been examined and compared with those of degraded phytochrome (118/114 kDa). The CD spectrum of the P_r form of 124 kDa phytochrome does not differ significantly in the visible region from that of 118/114 kDa P_r. In contrast, the CD spectrum of the P_fr form of 124 kDa phytochrome differs from that of the 118/114 kDa species in the far-red, red and blue regions of the spectrum. This result confirms that the NH₂-terminal polypeptide segment has a critical role in chromophore-protein interaction in the P_fr but not in the P_r form. In the UV region, 124 kDa phytochrome exhibits a photoreversible difference between the CD spectra of P_r and P_fr, whereas no such difference is observed for 118/114 kDa preparations. These data suggest a possible photoreversible change in secondary structure of the 124 kDa phytochrome polypeptide that requires the presence of the 6/10 kDa NH₂-terminal domain to occur.     

DIFFERENTIAL EFFECT OF CALCIUM ON CHLOROPLAST MOVEMENT IN MOUGEOTIA

The flat, ribbon-shaped chloroplast in the filamentous green alga Mougeotia sp. undergoes light-induced orientational movement controlled by an intracellular tetrapolar gradient of the active form of phytochrome, P_fr. Some substructural and physiological aspects of this reaction were studied. An intracellular pattern of microfilaments (diameter: 5–10nm), presumably related to chloroplast movement, was identified in situ. In addition, it could be shown that chloroplast movement decreases parallel to a nitric acid soluble fraction of intracellular calcium. These results might indicate that phytochrome governs chloroplast movement in Mougeotia via control of the binding state of calcium.     

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.     

QUANTITATIVE CORRELATION BETWEEN SPECTROPHOTOMETRIC PHYTOCHROME ASSAY AND PHYSIOLOGICAL RESPONSE

Abstract— Apparent synthesis* of the enzyme lipoxygenase in the cotyledons of the mustard seedling (Sinapis alba L.) is controlled by phytochrome (P_{fr ground state})? through a threshold (all-or-none) mechanism. This response was used to determine physiologically the photostationary states, Λ that is, the [P_fr]/[P_tot] ratios established by different wavelengths in the red and far-red range of the spectrum, including the standard red and far-red sources used in this laboratory (Mohr, 1966). Under the premises (for which justification has been given on previous occasions) that the [P_fr]/[P_tot] ratio for standard red light is 0.8, and that the decay of P_fr is a first-order process with a half-life of 45 min, the [P_fr]/[P_tot] ratios determined physiologically by means of the lipoxygenase response agree with the [P_fr]/[P_tot] ratios determined spectrophotometrically by Hartmann and Spruit (cf. Fig. 9 in Hanke et al., 1969) in hypocotyl hooks of mustard seedlings. In the hook the _fr, that is, the [P_fr]/[P_tot] ratio for standard far-red, is found to be 0.023. In the cotyledons, this ratio is several times higher (Schafer et al., 1972). The conclusion that apparent lipoxygenase synthesis in the cotyledons is controlled by phytochrome located in the hook has been substantiated by further spectrophotometric (Schäfer et al., 1973) and physiological experiments (H. Oelze-Karow and H. Mohr, in preparation). The minimum steepness of the threshold was determined. An increase of the P_fr level from 118 (relative units) to 130 leads to an instantaneous and total suppression of apparent lipoxygenase synthesis; a corresponding decrease from 130 (relative units) to 118 leads to an immediate resumption of apparent LOG synthesis at full speed. It is concluded that an explanation of the experimental facts requires a cooperative effect on the level of P_fr, a high degree of synchrony on the cellular and organismic level and rapid communication between the hypocotyl hook and the cotyledons. *The term ‘apparent synthesis’ is used operationally in the present paper to denote any increase of enzyme activity, although de novo synthesis of lipoxygenase has not so far been rigorously demonstrated. The usual inhibitor experiments (cf. Oelze-Karow et al., 1970) have led to the conclusion that intact RNA and protein synthesis is required for an increase of lipoxygenase activity.     

ANALYSIS OF Pfr DESTRUCTION IN AMARANTHUS CAUDATUS L EVIDENCE FOR TWO POOLS OF PHYTOCHROME*

Abstract— Kinetics of the destruction of the far red absorbing form of phytochrome (P_fr), measured by in vivo spectroscopy, show two phases: after a saturating red light pulse, rapid first order decay results in the loss of most, but not all, of the detectable P_rr; decay of the rest is much slower. The concentration of the more stable P_fr is positively correlated to the concentration of the total P_fr established at time zero. The linear relationship between total and ‘stable’ P_fr exludes the existence of a threshold level of P_fr for fast destruction. Photoconversion of the P_r (red absorbing form of phytochrome) present during the slow decay, by exposure to a second light pulse, is followed by fast destruction of most of the newly formed P,_r, whereas some P_fr formed by the first pulse still remains. The experiment suggests that not all P_fr molecules are accessible to the same destruction mechanism, i.e. there are two populations of P_fI.     

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.     

PHYTOCHROME IN ETIOPLASTS IN RELATION TO CHLOROPHYLL ACCUMULATION*

Abstract— De-etiolation of maize seedlings reduces their sensitivity for red light potentiation of rapid chlorophyll accumulation in white light. An earlier proposal (Raven and Spruit, 1973) attributes this to migration of the far-red absorbing form of phytochrome (P_fr) to receptors essential for chlorophyll synthesis, thereby increasing the local P_fr/total phytochrome (P_tot)ratio. We have studied etioplasts as possible loci for such P_(r receptors. The level of spectrophotometric phytochrome in purified etioplasts isolated from red preirradiated maize seedlings was higher than that of dark grown plants. The difference was marginally significant, however. We argue that migration of a fraction of cytoplasmic P_fr to the etioplasts, too small to be spectrophotometically demonstrable, could still meet the requirements of the model. Dark destruction of bulk spectrophotometric P_fr following saturating red irradiation of seedlings is not paralleled by a decrease of etioplast phytochrome. the latter remaining essentially constant over long periods. On the other hand, the potentiating effect of red light in intact seedlings is still partially reversible by far red light even after 24 h of darkness when destruction of bulk P_fr is complete. Since this demonstrates persistent presence of P_fr active in potentiation, we propose that at least part of this P_fr is associated with the etioplasts.     

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.     

AN ATTEMPT TO LOCALIZE THE THRESHOLD REACTION IN PHYTOCHROME-MEDIATED CONTROL OF LIPOXYGENASE SYNTHESIS IN THE MUSTARD SEEDLING

Abstract —Synthesis* of the enzyme lipoxygenase (LOG)? in the cotyledons of the mustard seedling (Sinapis alba L.) is controlled by phytochrome (P_fr) through a threshold (all-or-none) mechanism. The data of the present paper confirm the previous assumption (Oelze-Karow and Mohr, 1973) that the primary reaction of P_fr (P_fr+ X → P_frX ? P_frX‘) is the site of the highly cooperative threshold reaction. Suppression of LOG synthesis depends on the presence of P_frX’. However, P_frX‘ is only stable above the threshold level of P_fr. If the level of P_fr is below the threshold, P_frX is stable, and no suppression of LOG synthesis occurs. As long as the level of P_fr remains below the threshold, no destruction of P_fr takes place. Destruction of P_fr occurs only as long as [P_fr]?is above the threshold level. Thus the simplest formulation of the actual threshold reaction in the LOG response is P_frX?_frX’ state at [P_fr] below threshold no P_fr destruction LOG synthesis suppressed state at [P_fr] above threshold P_fr destruction(¹k_d LOG synthesis unimpaired The reversible threshold reaction is thus an integral part of the “primary reaction” of P_fr occurring at the “matrix” specific for the LOG response. The data and conclusions on the LOG response are consistent with an “open phytochrome-receptor model” recently advanced by E. Schäfer (1975). The data are not consistent with the concept that a rapid dark reversion (P_fr→P_r) exists in dicotyledonous seedlings and that the degree of P_fr dark reversion strongly depends on the initial photostationary state, φ?, established by a saturating light pulse.     

The Complexity of the Pr to Pfr Phototransformation Kinetics Is an Intrinsic Property of Native Phytochrome*

Several possible origins of the complex phytochrome red to far-red light-absorbing phytochrome (P_r P_fr) phototransformation kinetics in the nanosecond-to-second time range have been examined. Heterogeneity based on protein sequence is ruled out as an origin of the multi-component kinetics because recombinant 124 kDa oat phytochrome A apoprotein reconstituted with phytochro-mobilin and the native protein are very similar in this regard throughout this time range. The P_r forms of native 124 kDa oat phytochrome A and of a homogeneous recombinant 65 kDa chromoprotein fragment exhibit thermochromic properties interpreted as arising in each case from the presence of two P_r species in thermal equilibrium. They exhibit identical photochemical properties. The complex kinetics therefore cannot result from P_r heterogeneity either. Thus, the presence of two P_r forms in equilibrium (P_r,₆₇₅ and P_r,₆₅₅) and the complex multiex-ponential P_rP_fr phototransformation kinetics observed in all time ranges are intrinsic properties of the homogeneous holoprotein of oat phytochrome A.     

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.