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.     

Chromophore structure in phytochrome intermediates and bleached forms of phytochrome

Phytochrome (120 kdalton or 60 kdalton) was isolated from etiolated seedlings of Avena sativa L. cv. Pirol (Baywa München). Irradiation with red light of the P_r form at −23°C in aqueous medium or at −40°C in 66% glycerol leads to the intermediate meta-Rb. Acidification of the glycerol solution at −40°C leads to the absorption of the 15(E) phytochrome chromophore (= P_fr chromophore). Subsequent irradiation transforms this into the 15(Z) chromophore (= P_r chromophore). The presence of the 15(E) chromophore was demonstrated by the same methods also in phytochrome bleached either as P_fr in the dark by 4 M urea, methanol, acetone, ethylene glycol, 8-anilinonaphthalene-1-sulfonate, or as P_r by irradiation with red light in the presence of the same agents. Phytochrome bleached by sodium dodecylsulfate or by dehydration was also investigated. It was concluded that bleached phytochrome contains the P_fr chromophore without specific interaction with the protein.     

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.     

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.     

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°.     

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.     

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.     

Action spectra for chlorophyll formation during greening of wheat leaves in continuous light

The contents of Chl a and Chl b in dark-grown leaves of wheat ( Triticum aestivum L. cv. Starke II, Weibull) irradiated for 20 h with light of different wavelengths and intensities have been measured. The amount of Chl b formed revealed a higher maximum in the Chl b /Chl a ratio for light most strongly absorbed by the phytochrome system and giving a high [P_fr]/[P_tot] ratio. This indicates a stronger enhancing effect of P_fr on Chl b formation than on Chl a formation. Similarities between action spectra for Chl a and b formation indicate that Chl b originates from Chl a . A comparatively high effectiveness of light within the wavelength range 500–550 nm as compared to the action spectrum for PChl→Chl phototransformation shows that PChl₆₃₆, in addition to PChl₆₅₀, takes part in the greening process.     

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.     

Photophobic stop-response in a dinoflagellate: Modulation by preirradiation

Gyrodinium dorsum Kofoid responds photophobically to flashes of blue light. The photophobic response consists of a cessation of movement (stop-response). Without background light and after a flash fluence above 10 J m⁻², 75–85% of the cells show a stop-response, while only 50% of the cells show this response at 5 J m⁻². With a flash fluence of 5 J m⁻², background light of different wavelengths either increases (614 nm. 5.5–18.2 μmol m⁻² s⁻¹) or decreases (700 nm, 18.4–36.0 μmol m⁻² s⁻¹) the stop-response. Two hypotheses for the mechanism of the modulation by background light of the photophobic response are discussed: an effect of light on the balance of the photosynthetic system (PS I/PS II) or an effect on a phytochrome-like pigment (P_r/P_fr). This study supports the idea that a phytochrome-like pigment works in combination with a blue light-absorbing pigment. It was also found that cells of Gyrodinium dorsum cultured in red light (39.8 μmol m⁻²) had a higher absorption in the red region of the absorption spectra than those cultured in white light (92.7 μmol m⁻²).     

Involvement of ethylene in the abscission of flowers and petals of Leptospermum scoparium

The growth of cotyledons and primary leaves of I-day-old Sinapis alba L. plants were studied under various light conditions and action spectra produced. For both responses blue and red light are most effective and a strong fluence rate dependency can be observed. The red light effect appears to be mediated through phytochrome, that of blue light being due to a separate blue light receptor, although this receptor requires the presence of far-red absorbing phytochrome (P_fr) in order to be effective.     

Capacity of cytochrome and alternative path in coupled and uncoupled root respiration of Pisum and Plantago

A critical duration of darkness must be exceeded for the photoperiodic induction of flowering in short-day plants. This requires detection of the light/dark transition at dusk and the coupling of this information to a time-measuring system.
Lowering the P_fr/P_tot, ratio photochemically at the end of the day did not accelerate the onset of dark timing in Pharbitis nil Choisy cv. Violet. Time-measurement was initiated when, with no change in spectral quality, the irradiance fell below a threshold value. Thus, if the light/dark transition at dusk is sensed by a reduction in P_fr, this reduction can be achieved as rapidly through thermal reactions as through photochemical ones. When given at hourly intervals during a 6-h extension of a 24-h main light period in white light, pulses of red light were as effective as continuous red light in delaying the onset of timing; pulses every 2 or 3 h were less effective. The effectiveness of intermittent red light indicates that phytochrome is the photoreceptor and the requirement for frequent exposures suggests that P_fr is lost rapidly in the dark. However, the red light pulses could not be reversed by far-red light, which argues against this hypothesis. An alternative explanation is that the perception of light as being continuous occurs only when "new" P_fr is regenerated sufficiently frequently.
The nature of the coupling of the dusk signal to the time-measuring system is discussed and it is suggested that the effect of each red light pulse is to delay the phase of the photoperiodic rhythm by 1–3 h.     

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.     

Enhancement of Pfr-mediated responses by light pretreatments: persistence of the pretreatment effects

Abstract. In germinating seedlings of Sinapis alba , nitrate reductase activity is under phytochrome control and becomes accessible to phytochrome at about 15 h from sowing. The induction of the enzyme with pulses of light is strongly affected by pretreatments given prior to 15 h, also acting through phytochrome. It is shown that the effects of these pretreatments can persist undiminished for a considerable time (>40 h) but do not alter the pattern of the subsequent responsiveness to P_fr. The nitrate reductase response is compared with other data pertaining to a similar response.     

Light-quality effects on Arabidopsis development. Red, blue and far-red regulation of flowering and morphology

Arabidopsis thaliana (L.) Heynh. race Columbia plants were grown in red. blue, red + far-red, blue + far-red and various light mixtures of red + blue + far-red light under 14 h light/10 h dark photoperiods. Each single light source and light mixture maintained a constant irradiance (50 μmol m⁻² s⁻¹) and the mixtures of red + blue + far-red maintained a constant ratio of red/far-red light, but varied in the ratio of blue to red + far-red light. Depending on the method used for calculation, values of the fraction of phytochrome in the far-red absorbing form (P_fr/P_tot) for these light mixtures were either constant or decreased slightly with increasing percentage of blue light in the mixtures. Arabidopsis flowered early (20 days) in blue, blue + far-red and red + far-red light and late (55 days) in red light. In mixtures of red + blue + far-red light, each of which established a nearly constant P_fr/P_tot flowering was in direct relation to time and irradiance level of blue light. Leaf area and petiole length were also correlated with blue light irradiance levels.     

Endurance at intermediate swimming speeds of Atlantic mackerel, Scomber scombrus L., herring, Clupea harengus L., and saithe, Pollachius virens L.

Endurance and swimming speed were measured in mackerel, herring and saithe when they were induced by the optomotor response to swim at prolonged speeds along a 28-m circular track through still water in a 10-m diameter gantry tank. The maximum sustained swimming speed ( U _ms was measured as body lengths per second ( b.l.s ⁻¹) for each species and for saithe of different size groups. Herring with U _ms of 4.06 b.l.s ⁻¹ (25.3 cm, 13.5°C) were the fastest, mackerel U _ms was 3.5 b.l.s ¹ (33 cm, 11.7°C) and saithe (14.4°C) showed a size effect where U _ms at 25 cm was 3.5 b.l.s ¹ and at 50 cm 2.2 b.l.s ¹. When swimming at speeds higher that U _ms, all three species showed reduced endurance as speed increased. How the curved track reduces the swimming speed is discussed.     

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.