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.     

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.     

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.     

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.     

The effect of SAN 9789 and light on the germination of seeds from Taraxacum vulgare

Photoblastic seeds (achenes) of Taraxacum vulgare coll. were treated with a water solution of SAN 9789, 4-chloro-5 (methylamino) -2- (α,α,α-trifluoro- m -tolyl) -3(2H) pyridazinone. SAN-treatment increased the germination in darkness from 0 to 12%. An irradiation for 5 min with red light, giving a germination of 12% for seeds in water only, gave together with SAN treatment a germination of 60%. In both water and SAN, the effect of red irradiation could be reversed by a short irradiation (15 min) of far-red light. If far-red light was repeatedly given (5 min per h) it had hardly any effect on germination in water (4% germination), but for seeds in SAN solution, intermittent far-red light had a stimulating effect (63% germination). If far-red light was given continuously for 96 h, the germination in water was 1% and in SAN solution 17%. The results in the present paper indicate that SAN may broaden the concentration interval of P_fr for which germination is high.     

Photoregulation of seed germination of wild-type and of an aurea-mutant of tomato

Seed germination of an aurea mutant of tomato ( Lycopersicon esculentum Mill.) is promoted by continuous irradiation with red, far-red or long-wavelength far-red (758 nm) light as well as by cyclic irradiations (5 min red or 5 min far-red/25 min darkness). Far-red light applied immediately after each red does not change the germination behaviour. Seed germination of the isogenic wild-type, cv. UC-105, is promoted by continuous and cyclic red light while it is inhibited by continuous and cyclic far-red light and by continious 758 nm irradiation. Far-red irradiation reverses almost completely the promoting effect of red light. The promoting effect (in the aurea mutant) and the inhibitory effect (in the wild-type) of continuous far-red light do not show photon fluence rate dependency above 20 nmol m⁻² s⁻¹. It is concluded that phytochrome controls tomato seed germination throgh low energy responses in both the wild type and the au mutant. The promoting effect of continuous and cyclic far-red light in the au mutant can be attributed to a greater sensitivity to P_fr.     

Synergism between gibberellic acid and low Pfr levels inducing germination of Kalanchoë seeds

Sown on water, seeds of Kalanchoëbiossfetdiana Poelln. cv. Feuerblute are absolutely light-requiring and show full red/far-red reversibility. In seeds, sown on 2 ×10^-3 M gibberellic acid, red/far-red reversibility disappears and both short red and far-red irradiations induce germination. Gibberellic acid alone does not induce germination, but it increases the physiological activity of P_fr to the extent, that the low P_fr level obtained by far-red irradiation becomes very effective. The synergism between gibberellic acid and far-red light appears after a two-day incubation; period. The nature of this lag phase was examined by measuring both germination and uptake of labelled gibberellic acid in intact seeds and seeds with a punctured seed coat. The lag phase was shown to be independent of the uptake kinetics of gibberellic acid and allows development to a specific stage, necessary for germination after phytochrome-phototransformation. The kinetics of the uptake of gibberellic acid by intact seeds and embryos of intact seeds are different. In intact seeds most of the gibberellic acid is retained in the seed coat; only a small fraction actually penetrates to the embryo where it can exert its physiological activity.     

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.     

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.     

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.     

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.     

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°.     

Gibberellin-biosynthesis and -sensitivity mediated stimulation of seed germination of Sisymbrium officinale by red light and nitrate

Light stimulated seed germination of Sisymbrium officinale (L.) Scop, (hedge mustard) by means of two different mechanisms. Light effect I was absolutely dependent on the simultaneous presence of nitrate. Without nitrate, red (R) irradiated seeds did not escape from the antagonizing action of far-red (FR) irradiation. The data indicated that nitrate acted as a cofactor at the level of the FR absorbing form of phy-tochrome (P_fr). The combined action of R and nitrate could be replaced by addition of the gibberellins 4 and 7 (GA,₄₊₇). This action could be inhibited by the growth re-tardant tetcyclacis, an inhibitor to GA biosynthesis in cell free systems and intact plants. The action of tetcyclacis was fully neutralized by GA₄₊₇. It is concluded that the combination of R and nitrate stimulated GA biosynthesis. Omission of nitrate from the incubation medium enabled the study of light effects apart from G A biosynthesis. In such conditions R stimulated the sensitivity to GA₄₊₇, (light effect II). The two light effects could also be distinguished by their different reactions to the temperature of a pre-treatment in water and darkness. The sensitivity to R and nitrate was subject to breaking and induction of dormancy. Both processes were stimulated at rising temperatures. Due to a different optimum, breaking of dormancy prevailed at lower temperatures and induction of secondary dormancy at more elevated temperatures. The sensitivity to GA₄₊₇ rose and fell in a comparable way during dark incubation at a broad range of temperatures. The capacity of light to stimulate GA₄₊₇, action did not diminish at higher temperatures, it even tended to rise. The study indicated that seed germination is regulated by an increase in both the levels of GAs and the sensitivity to GAs.     

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.     

Light and temperature action in germination of seeds of the empress tree (Paulownia tomentosa)

Seeds of the empress tree ( Paulownia tomentosa Steud.) were imbibed for two weeks in darkness at constant temperatures (18, 23 or 28°C), and then irradiated with red light for 5 min. Germination was poor if it took place at the same temperature as imbibition, but a high percentage was achieved if the seeds were exposed to higher or lower temperatures before they were irradiated. Maximum germination was obtained when the difference between pretreatment and imbibition was about 10°C. The effect increased with the duration of the pretreatment and was optimal at 24 h. The effect decreased as the time lapse between temperature pretreatment and red light irradiation increased, and it was lost after two days. If pretreatment was shorter than 24 h (12 h). a high percent of germination was obtained by alternating pretreatment and imbibition temperatures. The germination of seeds imbibed in 40% heavy water was also stimulated by temperature pretreatments. Light and temperature also exhibited an interactive effect in the germination of seeds that were imbibed in darkness for only 3 days. For each of the germination phases there was a temperature at which the time needed for 50% germination was the shortest, namely 35°C during imbibition, 37.5°C in the period of P_fr activity. and 32.5°C during radicle protrusion. The data obtained are shortly discussed in relation to the domestication of empress tree in Southern Europe.     

Photocontrol of seed germination in Impatiens wallerana Hook. f.

General characteristics of light sensitivity of Impatients wallerana seeds were investigated. Germination was absolutely dependent on light, irrespective of temperature. High percentages of germination were obtained by exposure to long periods of illumination or, alternatively, to several repeated short irradiations with red light. In this case, responsiveness to light was not altered by increasing either the initial incubation period in darkness or the dark intervals between short exposures. Effects of red light were reversed by far-red light, thus demonstrating the involvement of phytochrome. Evidence was presented for an interactive effect, of unknown physiological nature between red and far-red light on the germination of the seeds.Abbreviations P_r phytochrome, red light absorbing form - Pfr phytochrome far-red absorbing form     

Mathematical approach to effects of repeated treatments in the study of very low fluence, low fluence and high fluence germination responses

Theoretical calculation of the germination response induced by repeated treatments, separated by a dark period long enough to enable fixation of the effect of the preceding treatment, is possible when defining the percentage germination induced by the first treatment as the responding proportion (p) of the total treated seed population. Consequently the germination response induced by a second treatment should be relative to the proportion (q) of the seed population not responding to the first treatment (q = 1 - p).
The fitting of these calculations with experimental data for the Very Low Fluence Response (VLFR) for germination of seeds of Kalanchoë blossfeldiana Poelln. cv. Vesuv, induced by repeated light pulses, suggests the independency of the effect of each treatment, i.e. the effect of the second treatment is neither positively nor negatively influenced by the first treatment.
This hypothesis is not valid for calculation of the Low Fluence Response (LFR) for germination of Kalanchoë seeds induced by repeated light pulses, since the first light pulse does not result in a germination response. At least two irradiations are needed for an LFR while the third and following pulses increase the response much more than calculated with the proposed equation. It is suggested that the LFR in Kalanchoë , in contrast to the VLFR, includes the involvement of some pre-existing far-red absorbing form of phytochrome (P_fr) and the involvement of dark reactions are to be considered.
The effect of long irradiation times (up to 2xl0⁵ s) resulting in a (defined in this paper) high fluence response (HFR) for germination of Kalanchoë seeds is also discussed in terms of independently responding seed population fractions.     

An unusual effect of the far-red absorbing form of phytochrome: Photoinhibition of seed germination inBromus sterilis L.

Seeds ofBromus sterilis L. germinated between 80–100% in darkness at 15° C but were inhibited by exposure to white or red light for 8 h per day. Exposure to far-red light resulted in germination similar to, or less than, that of seeds maintained in darkness. Germination is not permanently inhibited by light as seeds attain maximal germination when transferred back to darkness. Germination can be markedly delayed by exposure to a single pulse of red light following 4 h inhibition in darkness. The effect of the red light can be reversed by a single pulse of far-red light indicating that the photoreversible pigment phytochrome is involved in the response. The response ofB. sterilis seeds to light appears to be unique; the far-red-absorbing form of phytochrome (Pfr) actually inhibiting germination.Abbreviations Pr red absorbing form of phytochrome - Pfr far-red absorbing form of phytochrome     

After-ripening and phytochrome action in seeds of dormant lines of wild oat (Avena fatua)

The effects of a short exposure to red, far-red or alternate red/far-red light on the germination of seeds after-ripened for different periods of time were studied in dormant lines of wild oat ( Avena fatua L.). Three stages were distinguishable in the after-ripening period in the response of germination to light. Seeds stayed dormant and showed no response to light during stage I. Phytochrome-mediated germination was observed in seeds during stage II. The phytochrome action disappeared during stage III, i.e. seeds fully germinated following treatments of all light qualities. When the seeds were imbibed in polyethylene glycol solutions, dark germination was reduced and phytochrome again had an effect, which suggested the involvement of phytochrome in water uptake of the seed.     

The effect of light and exogenous gibberellic acid on respiration pathways during germination of tomato seeds

Tomato (Lycopersicon esculenlum Mill. cv. Taiwan Red) seeds are typical lightsensitive seeds where light requirement can be substituted by gibberellic acid (GA₃). During the initial stage of germination, the hexose monophosphate pathway (HMP) in seeds incubated for 24 h under white light or 12 h in 0.3 mW GA₃ solution acidified with 1 M HCI for 1 h in the dark (HCl→GA₃) was much greater than in seeds incubated for 24 h in the dark. The results were obtained from measurements of the respiration rate by man metric method of Warburg with or without iodoacetic acid, and from activities of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and aldolase (EC 4.1.2.13). Although the HMP tended to decrease at the later stage (72 h under white light or in the dark, or 36 h in HCl→GA₃), it remained greater in light- than in dark-incubated seeds. CN-resistant respiration increased from 27–30% to 57–64% of the total respiration in seeds incubated under white light or in HCI→GA₃, while respiration of non-germinating, dark-incubated seeds remained zero. Benzohydrox–amic acid (BHAM), an inhibitor of the alternative pathway, inhibited both respiration and seed germination. It is concluded that the enhancement of HMP and the CN–resistant pathways are both controlled by P_fr, but there is no direct connection between them.