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.     

PHYTOCHROME CONTROL OF ITS OWN SYNTHESIS IN Sorghum vulgare AND Avena sativa

The accumulation of phytochrome in the dark was measured for Avena sativa seedlings after a white light pretreatment and for Sorghum vulgare seedlings after continuous red or far-red light treatments, using the herbicide Norflurazon to prevent greening under continuous irradiation. In both cases the accumulation of phytochrome depends on the state of the phytochrome at the light-dark transition: high P_fr levels (red light pulse) led to a slower rate of phytochrome accumulation than lower P_fr levels (long wavelength far-red (RG 9) light pulse). Poly-(A⁺)-RNA was isolated fromA. sativa seedlings grown for 48 h in darkness + 24 h WL + light pulse (5 min) (red, RG 9 light, red followed by RG 9 light or RG 9 followed by red light pulse) + 19 h darkness. The poly-(A⁺)-RNA was translated in a rabbit reticulocyte lysate system and the translation products were immunoprecipitated by specific anti-phytochrome antibodies. It was demonstrated that the activity of mRNA coding for phytochrome was under phytochrome control.     

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.     

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.     

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.     

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.     

SPECTRAL PROPERTIES,PHOTOTRANSFORMATION KINETICS AND PELLETABILITY OF PHYTOCHROME IN Pharbitis nil Chois*

Abstract— An examination has been made of the involvement of phytochrome in the circadian rhythm of flowering in Pharbitis nil Chois. The peak position of P_fr absorption changes with time after a red light pulse. The shortest absorption wavelength of P_fr occurs at the same time as flowering is inhibited by red light in dark grown, red light pretreated plants. Pelletable and supernatant phytochrome show a similar trend with lowest values found at the time of flower inhibition. Neither phototransformation kinetics nor intermediates of phytochrome which accumulate in white light show such a relationship to the circadian rhythm found in flowering of dark grown P. nil.     

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.     

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.     

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.     

PHYTOCHROME ACTION: A REAPPRAISAL

Stems of fully green plants show at least two types of response to light. In one, P_fr inhibits elongation. The second is a promotion of elongation which operates only in light; the effectiveness of red and far-red wavelengths indicates that this response is also mediated through phytochrome. Studies of the de-etiolation process also provide evidence for two modes of action of phytochrome; one is a P_fr-dependent reaction, and the second requires continuous light (or frequent short irradiations). It is proposed that, in addition to reactions which require P_fr and proceed in darkness, an important reaction of phytochrome in green plants occurs only in light. We have termed these two modes of action of phytochrome “static” and “dynamic”. The static mode operates after a brief exposure to light which establishes P_fr; the potential responses are largely reversible by far-red and exhibit reciprocity. The dynamic mode operates only in light and the responses do not show reciprocity. We consider that this mode operates through the transition from one bound form of phytochrome to another. The possible involvement of these two modes of action of phytochrome in photoperiodic mechanisms is discussed.     

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.     

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.     

A METHOD FOR MEASURING PHYTOCHROME IN PLANTS GROWN IN WHITE LIGHT

Abstract— Quantitative spectrophotometric measurement of phytochrome in plants grown for relatively long periods of time in white light is not possible due to the presence of chlorophyll. A method is described that prevents the accumulation of chlorophyll permitting such direct spectrophotometric measurement in light-grown tissue. Oat seedlings grown in the presence of the herbicide San 9789† (Norflurazon) for 6 days in constant light have considerably less chlorophyll than etiolated seedlings exposed to 1 min of light. Phytochrome concentrations measured in vivo and in vitro in these herbicide-treated plants were found to be about 2% of the level in etiolated tissue.     

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.     

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

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

IRRADIATION-ENHANCED PHYTOCHROME PELLETABILITY IN AVENA: IN VIVO DEVELOPMENT OF A POTENTIAL TO PELLET AND THE ROLE OF Mg2+ IN ITS EXPRESSION IN VITRO

The enhanced phytochrome pelletability that results from in vivo irradiation of Avena shoots may be divided into two operationally defined sequential stages: the in vivo development of a “potential to pellet” and the “expression” of this potential in vitro. Kinetic studies confirm previous findings that the generation of this “potential to pellet” is a very rapid (complete in < 10 s, 25°C), genuinely intracellular process, itself photoreversibly induced by P_fr. In addition, it is shown that the sustained development of the “potential to pellet”, that proceeds in the dark at 0°C following a red pulse, requires P_fr continually in the cell over the entire development period. Far red light immediately terminates further development of the red-induced “potential” at any point during the development phase. No immediate reduction is observed, however, in that level of “potential pelletability” already attained at the time of the far red pulse. This indicates that the level of “potential pelletability” established in vivo is insensitive to the form of the pigment at extraction regardless of the level reached. “Expression” of the “potential to pellet” refers to the actual detection in homogenates of an enhanced physical association of phytochrome with pelletable material. Maximum “expression” requires the presence of a divalent cation in the medium during homogenization. Rapid posthomogenization addition of Mg²⁺ to Mg²⁺-free extracts sustains enhanced pelletability but with rapidly declining effectiveness over the fmt 1–2 min after extraction. The rate of decline is faster if the phytochrome is present as P_fr than as P_r in the homogenate. Neither these nor previous data permit a distinction to be made between (a) preservation by the cation of a pre-existing intracellular interaction, and (b) a Mg²⁺-mediated induction of an artifactual, in vitro association predetermined in the cell by a genuine phyto-chrome-controlled process. Various formalistic models are discussed in the context of these and other data.     

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 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     

A SIMPLE FIELD METHOD FOR MEASURING LIGHT QUALITY: SEASONAL CHANGES IN A TEMPERATE DECIDUOUS WOOD*

Abstract— Light was measured at 5 m intervals along a ground level transect through a mixed deciduous woodland in SE England. A simple method was developed to characterise the light environment, using measurements of photon fluence rate in the blue, photosynthetically active radiation (PAR) and near infrared regions of the spectrum. It was demonstrated that the two parameters, required to describe the state of the photoreceptor phytochrome the photostationary proportion of P_fr and the cycling rate, can be calculated from the quantities of PAR and IR light. These parameters as well as the amount of blue light were followed throughout the year along the woodland transect. The main seasonal changes are the fall and rise in the proportion of light which is PAR (and hence in the proportion of phytochrome as P_fr) associated with the appearance and disappearance of the green leaf canopy.