Low temperature spectroscopy of phytochrome: Pr, Pfr and intermediates

Phytochrome (120 kdalton) was isolated from etiolated seedlings of Avena sativa L. cv. Pirol (Baywa, München). Low temperature spectra between −17°C and −160°C are recorded for P_r, P_fr, and irradiated phytochrome samples. The temperature-dependence of the P_r and P_fr absorption spectra is described. Difference spectra of such temperature effects can erroneously be interpreted as difference spectra of intermediates. Probable absorption spectra of intermediates are calculated from the spectra of irradiated P_r or P_fr, respectively. The calculated spectral data are compared with published data on phytochrome intermediates.     

Phototransformation of oat phytochrome with millisecond and submillisecond flashes: The level of the photostationary Pfr/Ptot ratio is modified by photoreversible intermediates

Photoconversion of the red-absorbing form of phytochrome (P_r) to the far-red-absorbing form of phytochrome (P_fr) and vice versa has been measured spectrophotometrically at 10°C in immobilized and soluble phytochrome (118 kdalton), prepared from 5-day-old etiolated oat seedlings ( Avena saliva L. cv. Sol II). The photostationary equilibrium φ= P_frP_tot (with P_tot= total amount of phytochrome P_r+ P_fr) for red light depends on whether it is established by repetitive pulses (≥ 5 s) or by repetitive flashes (≥ 4 ms). In the wavelength region around 660 nm, a lower φ is reached with flashes as compared to that with pulses. This difference becomes negligible if the wavelength is shortened to the 600 nm region, and it also disappears if the fluence of each individual flash is reduced. In contrast, in long-wavelength red light and short-wavelength far-red light, a higher φ is reached with flashes than with pulses.
We relate the differences in φ for flash and pulse irradiation to photochromic systems between P_r and photoreversible intermediates in the phototransformation pathway P_r→ P_fr. Thus, light absorption by phytochrome intermediates can be limiting for the quantitative relationship between light signal and P_fr formed.     

Low temperature spectrophotometry of the phototransformation of Pfr to Pr in pelletable pea phytochrome

Manabe, K. 1987. Low temperature spectrophotometry of the phototransformation of P_fr to P_r, in pelletable pea phytochrome.
Low temperature spectrophotometry was used to study the phototransformation of P_fr to P_r in 1000–7000 g pelletable fractions extracted from dark grown pea ( Pisum sativum L. cv. Alaska) epicotyls which had been irradiated with red and then far-red light. At -170°C, far-red irradiation of the pelletable phytochrome which had been pre-irradiated with saturating fluence of red light before freezing caused formation of an intermediate (named I₆₆₀), the difference spectrum of which showed a marked ab-sorbance decrease at 740 nm and a concomitant small increase at about 660 nm. The inermediate I₆₆₀ was converted to another intermediate (I₆₆₀) when it was warmed above -80°C. The difference spectrum of this intermediate showed a positive peak at 670 nm. This intermediate was photoconverted to P_fr by red irradiation and also underwent dark reversion to P_fr at -60°C. I₆₆₀ formed P_r if the temperature was above -10°C. The basic features of the phytochrome intermediates resemble those obtained in vivo and in degraded purified phytochrome.     

Circular dichroism of phytochrome intermediates

Phototransformation P_t to P_fr was investigated with 124-kDa phytochrome from etiolated oat seedlings ( Avena sativa L. cv. Pirol) using circular dichroism spectroscopy at -110°C to +30°C. Using absorption spectra of the intermediates formed at the respective temperatures, circular dichroism spectra (300–800 nm) of pure intermediates were calculated.
The sign of the circular dichroic absorption bands changed upon formation of lumi-R, the primary photoproduct of P_r. This would be compatible with a Z→E isomerization taking place at this reaction step. The subsequent intermediates (meta-R_a and meta-R_c) as well as P_fr showed only small circular dichroism. Their absorption spectra were drastically shifted, but had similar spectral shapes. The results are discussed in terms of conformational changes of the phytochrome chromophore presumably taking place at the early steps of phototransformation P_r to P_fr.     

Light-induced linear dichroism in photoreversibly photochromic sensor pigments. – V. Reinterpretation of the experiments on in vivo action dichroism of phytochrome

Experiments by several authors on the effects of polarized light on phytochromemediated responses in fern gametophytes and in the green alga Mougeotia have earlier been interpreted as showing that the transition moment of phytochrome in the P_r form is parallel to the plasmalemma, but perpendicular to the plasmalemma for the P_fr form of phytochrome. It is now shown that the experimental results can be interpreted differently, and that they are also consistent with a chromophore rotation of about 30° (instead of 90°), as found for immobilized phytochrome molecules in vitro. Thus there is no evidence for a rotation of the whole phytochrome protein. For the gametophyte of Adiantum it is calculated that the P_r transition moment is inclined 17° to the plasmalemma, and the P_fr transition moment ca 50°, corresponding to an in vivo chromophore rotation of ca 33°; however, these values are very approximate.     

Spectral properties and chromophore rotation of phytochrome bound to substituted Sepharose

Brushite purified phytochrome from Avena sativa L. cv. Sol II was bound to phenyl Sepharose, octyl Sepharose, CNBr-activated Sepharose and to anti-phytochrome immunoglobulins immobilized on Sepharose. The spectral properties of phytochrome bound to anti-phytochrome immunoglobulins and to phenyl Sepharose were similar to phytochrome in solution. Phytochrome bound to CNBr-activated Sepharose or to octyl Sepharose showed reduced P_fr formation after red irradiation. The reversal to P_r with far-red light was only partial but a further increase at 667 nm took place slowly in the dark. A peak at 657 nm was seen in the difference spectrum between CNBr-activated Sepharose-bound phytochrome kept in darkness and the identical sample immediately after a far-red irradiation.
The change in linear dichroism at 660 nm and 730 nm, induced by plane polarized red or far-red light, was measured. It was computed that the long-wavelength transition moment of phytochrome had an average rotation angle of 31.5° or 180°–31.5°. The substrate used for immobilization had a limited effect on the rotation angle. Phytochrome immobilized on CNBr-activated Sepharose gave an angle of 27.8° and phytochrome immobilized on phenyl Sepharose gave an angle of 32.6°.     

Binding of 124-kilodalton oat phytochrome to liposomes and chloroplasts

Binding of the radio-iodinated 124-kDa oat ( Avena sativa L. cv. Garry) phytochrome to liposomes and chloroplasts was investigated as a model system in order to understand the molecular affinity of phytochrome toward cellular organelles in plants. The binding of intact (124 kDa) phytochrome to liposomes and chloroplasts is hydrophobic in nature, as in the case of the degraded (118/114 kDa) phytochrome, but electrostatic interactions play a greater role in the intact phytochrome. The physiologically active P_fr form of the intact phytochrome showed a binding preference over the inactive P_r form with neutral liposomes and chloroplasts. However, the P_fr form of intact phytochrome exhibits smaller binding preference than the P_fr form of degraded phytochrome over their respective P_r forms (see Kim, I.-S. and Song, P.-S. 1981, Biochemistry 20: 5482–5489, for degraded phytochrome binding). These results indicate that the 6/10 kDa N-terminus segment, which is lost in the degraded phytochrome, plays an important role in determining the protein surface properties of the intact phytochrome. A competitive binding study on phytochrome also suggested that the P_fr form had a greater binding affinity for chloroplasts than the P_r form. However, the physiological activity of the P_fr form may not be explained simply by the observed difference in binding affinity between the two forms of phytochrome.     

The joint action of phytochrome and alternating temperatures in the control of seed germination in Dactylis glomerata

The promoting effect of light and alternating temperatures on the germination of seeds of three contrasting populations of Dactylis glomerata L. was studied. Irradiation treatments using broad band low irradiance light sources resulted in red/far-red reversible effects, demonstrating the involvement of phytochrome in germination control. Reduction of germination by far-red below the level of a dark control indicated the presence of high pre-existing levels of the active form of phytochrome (P_fr) in some individuals. The capacity for dark germination at 21/11°C (12 h/12 h) was shown to be dependent on P_fr. Although some individuals were capable of germination at constant temperatures following red irradiation, in most seeds germination was dependent on the presence of P_fr and alternating temperatures. Some individuals responded to a single red irradiation, although a large proportion of seeds required high levels of P_fr to be maintained for long periods. Previously published dose response curves for alternating temperatures and a measured dark reversion time of 48 h for P_fr established by a single 60 min red irradiation, provided firm evidence of a direct correlation between the requirements for repeated irradiation and number of alternating temperature cycles.     

Phytochrome in Norflurazon-treated seedlings of Pharbitis nil

The properties of phytochrome have been measured by dual-wavelength spectropho-tometry in the cotyledons of the short-day plant Pharbitis nil Choisy cv. Violet, where it is known to play a role in flower induction. In plants de-etiolated by a single white light period (4 h or longer), destruction of the far-red absorbing form of phytochrome (P_fr) was twice as rapid as after 10 min red light. A small fraction of P_fr was stable. After de-etiolation by a period of white light (6 h or longer) the rapid decrease of P_fr during the first 30 min was accompanied by a rapid increase of the red absorbing form of phytochrome (P_fr). This rapid increase of P_fr is probably due to dark reversion. Long term synthesis of phytochrome was inhibited by the presence of P_fr. Phytochrome synthesised in darkness showed the etiolated-plant type characteristics and underwent rapid destruction upon photoconversion to P_fr. The stable P_fr identified here is possibly that pool of phytochrome associated with the long term promotive process in flower induction, and the rapidly reverting P_fr is that pool associated with the night break inhibition of flowering.     

Coaction between phytochrome and blue/UV light in anthocyanin synthesis in seedlings

Light-mediated mass production of blue/UV absorbing pigments, anthocyanin and/or other flavonoid compounds, can be considered an adaptive mechanism to protect a plant against high levels of short wavelength sunlight. Comparative studies of light-mediated formation of anthocyanin in seedlings of higher plants have been performed. As a result of Darwinian evolution, a seedling may be expected to form considerable amounts of pigment only when necessary and only to the extent required for protection ('economy principle'). The four species investigated with regard to light-mediated synthesis of anthocyanin in seedlings (mustard, milo, tomato, wheat), differ greatly with regard to their photoperception. Phytochrome is involved in the photoresponse in all cases. We conclude that the P_fr-mediated differential gene activation leading to anthocyanin synthesis is the core of the response. However, the different species differ greatly with regard to the red, blue and UV light dependent processes they perform in order to establish sensitivity towards phytochrome (P_fr), or to amplify sensitivity towards P_fr.     

Phytochrome control of skotodormancy release in Grand Rapids lettuce achenes

Light-requiring Grand Rapids lettuce ( Lactuca sativa L.) achenes develop skotodormancy when imbibed in darkness for 7 days at 25°C. Redried skotodormant achenes maintain this type of dormancy upon subsequent rehydration. At 25°C full germination of skotodormant achenes can be induced by continuous and intermittent red light illumination as well as by several brief red irradiations given daily. One brief (10 min) red light irradiation can partly break skotodormancy at 20°C, while at lower temperatures the same treatment results in full induction of germination. Phytochrome control of the release from skotodormancy is proven by a) the dependence of the germination response on the relative sequence of red and far-red light in cyclic irradiations, and b) the reversion of red action by subsequent far-red irradiation. The time course of germination of skotodormant achenes treated with intermittent red light depends upon the length of dark interval between the light pulses. Germination is considerably delayed compared to that of non-skotodormant ones, induced by a single brief red light treatment. This fact in combination with the requirement, over a long period of time, of P_fr action for full manifestation of germination, indicates that skotodormancy is a deeper form of dormancy. It is concluded that the germination of lettuce achenes may always be subjected to phytochrome control.     

Spore germination and phytochrome biosynthesis in the fern Lygodium japonicum as affected by gabaculine and cycloheximide

We investigated whether the gradual increase in phytochrome content in the fern Lygodium japonicum (Thunb.) Sw. during dark imbibition results from hydration or from biosynthesis of phytochrome. Addition of gabaculine or cycloheximide to the culture medium caused inhibitions of both red light-induced spore germination and of the appearance of phytochrome in the spores. Fifty percent inhibition of both red light-induced germination and of the appearance of phytochrome in the spores occurred at ca 10⁻7 M cycloheximide. Red light-induced germination and phytochrome appearance were markedly inhibited by 10⁻4 M and completely by 10⁻3 M gabaculine, but germination induced by gibberellic acid was unaffected. Phytochrome was not detected in spores after forced hydration. These results suggest that the increase in phytochrome during imbibition was mainly due to de novo synthesis of the phytochrome apoprotein and to synthesis of the chromophore and/or proteins required for phytochrome formation, rather than to hydration of preexisting phytochrome molecules.     

Differences in phytochrome action on the formation of carotenoids and quinones during chloroplast development in seedlings of barley (Hordeum vulgare)

The influence of phytochrome on the light induced formation of carotenoids and quinones was investigated using etiolated seedlings of barley ( Hordeum vulgare L. cv. Villa). The biosynthesis of both the quinones and the carotenoids was enhanced by active phytochrome, but the formation of the individual carotenoids and quinones was influenced by quite different thresholds. In both younger and older plants the biosynthesis of β-carotene, lutein and violaxanthin was promoted by a low P_fr threshold. The formation of plastoquinone-9, plastohydroquinone-9, α-tocoquinone and phylloquinone was also influenced by a low P_fr threshold. The biosynthesis of zeaxanthin and neoxanthin required a much higher amount of P_fr. Only antheraxanthin-likeα-tocopherol, desmethylphylloquinone and, in older leaves, α-tocoquinone exhibited a complete reversibility of the phytochrome action in their biosynthesis. The effect of phytochrome on the biosynthesis of carotenoids and quinones was different in seedlings of different age.     

Interaction between the effects of phytochrome and gibberellic acid on the senescence of Alstroemeria pelegrina leaves

Chlorophyll loss in the leaves of cut flowering branches of Alstroemeria pelegrina L. cv. Stajello, placed in water in darkness at 20°, was inhibited by irradiation with red light and by the inclusion of gibberellic acid (GA₃) in the water. The effects of red light were abolished when it was followed by far-red light. Effects of GA₃ and red light were additive over a range of GA₃ concentrations (0. 01–1 μ M ). Chlorophyll breakdown was increased by the inclusion of AMO-1618, ancymidol, or tetcyclasis in the water. The effect of these inhibitors of gibberellin synthesis was fully reversed by GA₃. The inhibition of chlorophyll breakdown by red light was absent when AMO-1618, ancymidol or tetcyclasis were included in the water. The results indicate that leaf yellowing is controlled by endogenous gibberellins and that the effect of phytochrome is mediated by gibberellin synthesis.     

Nitrate reductase in cotyledons of cucumber seedlings as affected by nitrate, phytochrome and calcium

Regulation by the active form of phytochrome (P_FR) and the effect of Ca²⁺ was examined with nitrate reductase (NR) in etiolated cucumber ( Cucumis sativus cv. Beilpuig). Nitrate reductase activity (NRA) was studied in excised cotyledons of cucumber seedlings grown in distilled water and in darkness for seven days at 24 ± 0.5°C. All experiments were performed in the dark and a dim green safelight was used during analyses. In etiolated cucumber cotyledons NRA was induced by nitrate and a brief irradiation (15 min) with red light (R) resulted in 62% increase in NRA. This effect was nullified when R was followed immediately by a brief (5 min) far-red light (FR). NRA also showed a semidian (12 h) rhythmicity. Both P_FR, and nitrate effects were age dependent. Calcium seemed to be involved since the phytochrome effect was only observed when calcium was supplied in the external solution. The effect of R on NRA depended on the period of calcium nitrate incubation. An external supply of calcium ionophore mimicked the effect of R and, if supplied to R-irradiated cotyledons, produced a higher NR level than that caused by R alone. This suggested that intracellular free calcium was involved.     

The kinetics of type 1 phytochrome in green,light-grown wheat (Triticum aestivum L.)

The kinetics of type 1 phytochrome were investigated in green, light-grown wheat. Phytochrome was measured by a quantitative sandwich enzyme-linked immunosorbent assay using monoclonal antibodies. The assay was capable of detecting down to 150 pg of phytochrome. In red light, rapid first-order destruction of the far-red-light-absorbing form of phytochrome (Pfr) with a half-life of 15 min was observed. Following white light terminated by red, phytochrome synthesis was delayed in darkness by about 15 h compared to plants given a terminal far-red treatment. Synthesis of the red-light-absorbing form of phytochrome (Pr) was zero-order in these experiments. Phytochrome synthesis in far-red light was approximately equal to synthesis in darkness in wheat although net destruction occurred in light-grown Avena sativa tissues in continuous far-red light, as has been reported for other monocotyledons. In wheat, destruction of Pfr apparently did not occur below a certain threshold level of Pfr or Pfr/total phytochrome. These results are consistent with an involvement of type 1 phytochrome in the photoperiodic control of flowering in wheat and other long-day plants.Abbreviations ELISA enzyme-linked immunosorbent assay - FR far-red light - HIR high-irradiance response - Pfr farred-light-absorbing form of phytochrome - Pr red-light-absorbing form of phytochrome - Ptot total phytochrome (Pr + Pfr) - R red light The authors wish to thank Prof. Daphne Vince-Prue (University of Reading) for many helpful discussions regarding this work. Hugh Carr-Smith was supported by a Science and Engineering Research Council studentship and Chris Plumpton by an Agricultural and Food Research Council (AFRC) studentship. B. Thomas and G. Butcher were supported by the AFRC.     

Spectroscopic properties and chromophore conformations of the photomorphogenic receptor: phytochrome.

Fluorescence lifetimes of 'large (mol. wt. 120,000) and 'small' (mol. wt. 60,000) phytochromes isolated from oat and rye seedlings grown in the dark have been measured at 199 K and 298 K. Phytochrome model compounds have also been studied by phase modulation fluorometrically at 77 K for comparison with lifetime data for phytochrome. It was found that the fluorescence lifetime of 'large' phytochrome was significantly shorter than that of 'small' phytochrome and its chromophore models. The phytochrome chromophore of Pr form has been analyzed by fluorescence polarization, CD, and molecular orbital methods. The fluorescence excitation polarization of 'small' phytochrome and the chromophore model in buffer/glycerol mixture (3 : 1, v/v) at 77 K is very hight (0.4) at the main absorption band and is negative (--0.1) and close to 0 in the near ultraviolet band, respectively. Analyses of the spectroscopic data suggest that the chromophore conformation of Pr and Pfr forms of phytochrome are essentially identical. The induced ellipticity of 'large' rye phytochrome in the blue and near ultraviolet regions was found to be significantly higher than that of 'small' phytochrome, indicating that the binding interaction between the phytochrome chromophore and apoprotein is much tighter in the former than in the latter. In addition, the excitation energy transfer does occur from Trp residue(s) to the chromophore in 'large' phytochrome but not in 'small' Pr. This illustrates one feature of the role played by the large molecular weight apoprotein in the binding site interactions and primary photoprocesses of Pr. Finally, a plausible model for the primary photoprocesses and the mechanism of phytochrome interactions triggered by the Pr leads to Pfr phototransformation have been proposed on the basis of the above results.     

Binding of phytochrome to plasma membranes in vivo

A microsomal fraction was prepared by differential centrifugation from the homogenate of dark grown shoots of oats ( Avena sativa L. cv. Sol II). Plasma membranes were prepared from the microsomal fraction by means of an aqueous polymer two phase partition method. The content of phytochrome in the microsomal fraction and the plasma membrane fraction, respectively, were studied after different irradiation treatments of the intact shoots. Red irradiation increased the content of phytochrome in both the microsomal and plasma membrane fraction, especially in the presence of Mg²⁺. The increase induced by red light was fully reversible by far-red light for the plasma membrane fraction both in the presence and absence of Mg²⁺, in contrast to the microsomal fraction where Mg²⁺ had to be omitted. KI treatment of the membranes destroyed the binding of phytochrome whereas agents such as KCI, EDTA, CaCl₂ and Triton X-100, did not have this effect, indicating that the phytochrome attachment to the membrane is hydrophobic. This in vivo binding resembles to a large extent the one obtained in vitro by Sundqvist and Widell (Physiol. Plant. 59: 35–41, 1983) even though some differences between the phytochrome species and the membrane side exposed probably occur; so that the present interaction between phytochrome and the plasma membrane does not necessarily reflect the interaction that leads to physiological responses, and there could be more than one type of interaction.     

Photoreversible absorption change and domain structure of phytochrome

Phytochrome is a proteinaceous pigment that acts as a photoreceptor for photomorphogenetic responses in plants. It exists as two stable absorbing forms, P_r and P_fr, which are interconvertible reversibly by irradiation with red or far-red light. The present review discusses (i) the primary and higher-order structures of phytochrome that permit the reversible photoreaction; (ii) the molecular properties which change accompanying the phototransformation; and (iii) the four-leaved shape model which has recently been proposed as a model of quaternary structure of phytochrome.     

Differences in the nature of thermodormancy and far-red dormancy in lettuce seeds

Lettuce seeds cv. Noran germinate at 23°C in light as well as in darkness. However dormancy can be induced either by a long exposure (24 h) to far-red radiation or by an exposure of 48–72 h to a temperature of 37°C. The difference in response of these two types of dormant seeds to conditions inducing germination indicate that in both types P_fr is inactivated, but that a dark process required for immediate action of P_fr does not proceed at 37°C as it does during far-red radiation.