Phytochrome and Seed Germination. I. Temperature Dependence and Relative P(FR) Levels in the Germination of Dark-germinating Tomato Seeds

Germination of the dark-germinating seeds of 3 varieties of tomato is controlled by the phytochrome system. Germination is inhibited by far red radiation and repromoted by red applied after far red. At low temperatures, 17 to 20°, a single, low energy far red irradiation is sufficient to inhibit germination in all 3 varieties. At higher temperatures far red is less effective in the inhibition of the germination of the tomato seeds. The phytochrome fraction present as P_FR in the dark-germinating seeds of the Ace variety is about 40% of the total phytochrome present.     

Photocontrol of Anthocyanin Synthesis: IV. Dose Dependence and Reciprocity Relationships in Anthocyanin Synthesis

Under continuous far red light, anthocyanin synthesis in young, dark-grown cabbage seedlings (Brassica oleracea cv. Red Acre) is irradiance-dependent and fails to follow the reciprocity (irradiance × time = constant) relationships. Under intermittent far red treatments extended over a prolonged period of time, anthocyanin synthesis becomes dose dependent, and reciprocity relationships are valid. Intermittent far red treatments with short dark intervals between successive irradiations are as effective as continuous treatments, if the total radiation doses applied with the two types of treatments are equal and are applied over equally long periods of time. The high effectiveness of inter-mittent treatments, the dose dependence, and the validity of the reciprocity relationships suggest that cycling between red-absorbing form of phytochrome and far red-absorbing form of phytochrome and the formation of electronically excited far red-absorbing form of phytochrome, or the involvement of a second photoreactive system, besides phytochrome, may play only a minor role in high irradiance reaction anthocyanin synthesis brought about by prolonged exposures to far red irradiation.     

The “High Irradiance Responses” of plant photomorphogenesis

Plants growing in the natural environment are exposed daily to prolonged periods of high intensity irradiation. Many plant photomorphogenic responses are fully expressed only under prolonged exposures to high irradiances of light. The intensive study of these responses, the “High Irradiance Responses” (HIR) of plant photomorphogenesis, which started about 20 years ago, has been essentially directed—so far—toward the identification of the HIR photoreceptor, or photoreceptors, a problem that has not been satisfactorily and definitively solved, as yet. There is a great deal of evidence in support of the hypothesis that phytochrome, the pigment mediating the redfar red reversible plant photo-responses to low fluences of light, is involved in the photocontrol of the HIR. It seems likely that phytochrome may be the only photomorphogenic receptor responsible for the photocontrol of HIR responses brought about by irradiation at wavelengths longer than 600 nm. Phytochrome is probably also involved in the photocontrol of the HIR effects brought about by irradiation in the 350 to 500 nm region of the spectrum, but it cannot be excluded that other photochemical systems may also be involved. From a theoretical point of view, it does not seem unreasonable that the final expression of an HIR response may involve an interaction between phytochrome and other photochemical systems, with phytochrome probably playing the primary role and being responsible for the control of the activity of the other systems. Numerous “phytochrome only” interpretations (models) of the HIR have been proposed. Some of them have been developed to a fairly high degree of elaboration and have allowed the prediction of at least some of the features of the HIR. These “models,” although not rigorously and completely tested yet, seem to provide a reasonable interpretation for the HIR effects displayed under prolonged far red irradiation and for those HIR responses for which far red is the most effective spectral region. However, they do not provide a satisfactory explanation for the HIR responses for which blue is the most or the only effective spectral region, nor for the high effectiveness of white light. But, in spite of these problems, the “phytochrome only” interpretations of the HIR can be considered more satisfactory than those based on an interaction between phytochrome and other photochemical systems, especially in relation to the fact that the identity of these other photochemical systems has not been defined yet.     

Phytochrome and seed germination. V. Changes of phytochrome content during the germination of cucumber seeds

Cucumber seeds are light-sensitive, dark-germinating seeds. Inhibition of germination can be induced by prolonged exposure to continuous or intermittent FR. The dark germination process and the response to FR are phytochrome controlled. Phytochrome can be detected in these seeds by differential spectrophotometry in vivo. Spectrophotometrically measurable phytochrome increases during dark germination. The rate of increase is temperature dependent. Light treatments which are inhibitory for germination result in phytochrome contents lower than those of the seeds germinating in darkness. Treatments which restore germination also restore phytochrome formation.     

Light-dependent anthocyanin synthesis: A model system for the study of plant photomorphogenesis

The biosynthesis of anthocyanins in plant tissues either requires light or is enhanced by it. Light-dependent anthocyanin synthesis has been extensively used as a model system for studies of the mechanism of photoregulation of plant development. Two components can be distinguished in the action of light on anthocyanin production. The first component is the red-far red reversible, phytochrome-mediated response induced by short irradiations; the amount of anthocyanin formed in response to a single, short irradiation is small. The second component is the response to prolonged exposures; the formation of large amounts of anthocyanin requires prolonged exposures to high fluence rates of visible and near-visible radiation (290 to 750 nm) and shows the typical properties of the “High Irradiance Reaction” (HIR) of plant photomorphogenesis. Phytochrome is involved in the photoregulation of the HIR response and is the only photoreceptor mediating the action of prolonged red and far red irradiations. The response to prolonged ultraviolet and blue radiation is probably mediated, at least in some systems, by two photoreceptors: phytochrome and cryptochrome, the latter being a specific ultraviolet-blue-light photoreceptor. The nature of the interaction between phytochrome and cryptochrome in the regulation of plant photomorphogenic responses is still unclear.     

Blue light induced inhibition of seed germination: The necessity of the fruit coats for the blue light response

The seeds (achenes) of Laportea bulbifera require a chilling to break their dormancy and are negatively photoblastic. Their germination is inhibited by both continuous blue light and continuous or prolonged far-red radiation. The germination of de-coated seeds, prepared by removing the fruit coats, however, was strongly inhibited by continuous far-red, but not by continuous blue light. Photoreversible germination by a brief irradiation with red light occurred when the chilled seeds were exposed to prolonged far-red light. These results suggest that far-red light may regulate the germination of L. bulbifera seeds through the phytochrome system which exists in the regions other than fruit coats and that the blue light reaction may be governed by other photoreceptor system(s).     

Effects of light on phytochrome in cauliflower curd

The effect of light on the phytochrome content of cauliflower (Brassica oleracea (L.) var. botrytis) curd was studied using in vivo spectrophotometry. It was found that light caused a rapid increase in phytochrome level whereas transfer to darkness caused a rapid loss, regardless of the amount of phytochrome initially present in the far red absorbing form. The amount of phytochrome detectable during continuous irradiation appears to be related to the photoequilibrium , and is thus controlled by phytochrome itself.Abbreviation Pr and Pfr red and far red absorbing forms of phytochrome, respectively     

Phytochrome and Seed Germination: VI. Phytochrome and Temperature Interaction in the Control of Cucumber Seed Germination

Phytochrome control of cucumber seed germination is temperature-dependent. A prolonged exposure to radiation from broad spectrum far red sources (Pfr/P = 0.05 to 0.07) prevents germination at temperatures below 20 C. Above 20 C there is no inhibition and it appears as if there is an escape from phytochrome control. However, radiation from a monochromatic, narrow band 730 nanometer source (Pfr/P < 0.02) inhibits germination at temperatures above 20 C. This result supports the idea that, even at high temperatures, Pfr is responsible for the activation of germination. After 4 days of exposure to far red, a short red irradiation is quite effective in promoting germination if temperatures during the dark incubation periods are maintained below 20 C; red becomes effective at temperatures above 20 C. Promotion of germination will take place at a temperature of 25 C or higher without red irradiation. Again, we have an apparent escape from phytochrome control at high temperatures. However, if higher temperatures are used for only short periods, 2 to 6 hours, in combination with short red irradiation, one can demonstrate that activation of germination at high temperatures is still dependent on phytochrome. Phytochrome is probably destroyed during prolonged exposure to far red. Thus, the subsequent short red irradiation establishes levels of Pfr which may not be sufficient to promote germination at low temperatures but are probably adequate at high temperatures.     

Action Spectrum for Interaction between Visible and Far-Red Light on Face Chloroplast Orientation in Mougeotia

The orientation of chloroplasts from profile to face position in Mougeotia can be controlled in two ways: by a typical phytochrome-mediated system or by continuous, simultaneous irradiation with far-red and visible light. In experiments with dichromatic irradiation of Mougeotia, the light conditions applied prevented the formation of a far-red-absorbing form of phytochrome gradient in the cell. An unpolarized background of far-red light and linearly polarized monochromatic light of different wavelengths and vibrating parallel to the cell axis, if given by themselves, were completely ineffective in producing any changes in chloroplast orientation. Given together, however, changes in chloroplast orientation were induced. The action spectrum for this interaction between constant far-red and variable visible light was maximal at 620 nanometers. The chloroplast response in these dichromatic light conditions required a prolonged duration of exposure to simultaneous continuous irradiation of high fluence energy. The vibrating plane of linearly polarized 620 nanometer light had no significant influence on interaction with far-red light in chloroplast movement. The results obtained are different from the typical low energy phytochrome-mediated chloroplast orientation. This new type of chloroplast photoresponse might be mediated by an unknown sensory pigment.     

Phytochrome-mediated flavone glycoside synthesis in cell suspension cultures of Petroselinum hortense after preirradiation with ultraviolet light

Summary Ultraviolet light was demonstrated to stimulate flavone glycoside synthesis in Petroselinum cell suspension cultures. The data presented suggest the involvement of phytochrome in this response: Flavone glycoside formation resulting from 1 h of ultraviolet irradiation was increased by subsequent continuous far-red light irradiation. However, the ultraviolet effect was reduced by a subsequent irradiation with 10 min of far-red. This far-red effect was fully reversed by a sub-sequent irradiation with 10 min of red. Red and far-red irradiations were ineffective without ultraviolet preirradiation. It is concluded that in this system ultraviolet irradiation is required in order to change the cells in such a way as to allow a physiological effectiveness of the phytochrome system.     

Light-grown plants of transgenic tobacco expressing an introduced oat phytochrome A gene under the control of a constitutive viral promoter exhibit persistent growth inhibition by far-red light

A comparison of the photoregulation of development has been made for etiolated and light-grown plants of wild-type (WT) tobacco (Nicotiana tabacun L.) and an isogenic transgenic line which expresses an introduced oat phytochrome gene (phyA) under the control of a constitutive viral promoter. Etiolated seedlings of both the WT and transgenic line showed irradiance-dependent inhibition of hypocotyl growth under continuous far-red (FR) light; transgenic seedlings showed a greater level of inhibition under a given fluence rate and this is considered to be the result of the heterologous phytochrome protein (PhyA) functioning in a compatible manner with the native etiolated phytochrome. Deetiolation of WT seedlings resulted in a loss of responsiveness to prolonged FR. Light-grown transgenic seedlings, however, continued to respond in an irradiance-dependent manner to prolonged FR and it is proposed that this is a specific function of the constitutive PhyA. Mature green plants of the WT and transgenic lines showed a qualitatively similar growth promotion to a brief end-of-day FR-treatment but this response was abolished in the transgenic plants under prolonged irradiation by this same FR source. Growth inhibition (McCormac et al. 1991, Planta 185, 162–170) and enhanced levels of nitrate-reductase activity under irradiance of low red:far-red ratio, as achieved by the FR-supplementation of white light, emphasised that the introduced PhyA was eliciting an aberrant mode of photoresponse compared with the normal phytochrome population of light-grown plants. Total levels of the oat-encoded phytochrome in the etiolated transgenic tobacco were shown to be influenced by the wavelength of continuous irradiation in a manner which was qualitatively similar to that seen for the native, etiolated tobacco phytochrome, and distinct from that seen in etiolated oat tissues. These results are discussed in terms of the proposal that the constitutive oat-PhyA pool in the transgenic plants leads to a persistence of a mode of response normally restricted to the situation in etiolated plants.Abbreviations FR far-red light - R red light - WL white light - WL + FR white light supplemented with FR - HIR high-irradiance response - PAR photosynthetically active radiation - Pr, Pfr R- and FR-absorbing forms of phytochrome - Ptot total phytochrome - phyA (PhyA) gene (encoded protein) for phytochrome - WT wild type This work was supported by an Agricultural and Food Research Council research grant to H.S. and A.M.; J.R. Cherry and R.D. Vierstra, (Department of Horticulture, University of Wisconsin-Madison, USA) are thanked for the provision of the transgenic tobacco line.     

Growth and Dormancy in Lunularia cruciata (L.) Dum.: III. THE WAVELENGTHS OF LIGHT EFFECTIVE IN PHOTOPERIODIC CONTROL

Growth and dormancy in Lunularia are controlled by daylength,short-day promoting active growth, long-day or light-break treatmentinducing dormancy. Light-breaks of red light are highly effectivein inducing dormancy, while irradiation with other wavebandsis much less inhibitory to growth. Far-red light given afterred irradiation causes substantial reversal of the red-lighteffect, suggesting strongly that phytochrome is involved inthe photoperiodic response mechanism of Lunularia. However,even short(15 sec.) exposures to far-red light alone cause significantgrowth inhibition, and it is considered possible that far-redirradiation also leads to the formation of some of the P 730form of phytochrome.     

A red-far red reversible effect on uptake of exogenous indoleacetic Acid in etiolated rice coleoptiles

The uptake and accumulation of exogenous indoleacetic acid-¹⁴C by intact rice coleoptiles were examined. The absorption of exogenous indoleacetic acid was controlled by phytochrome, while the subsequent accumulation of this indoleacetic acid in various portions of the coleoptile was complex, and the effect of red light in this system was small compared to the alteration of the uptake of indoleacetic acid by red light. The absorption of indoleacetic acid exhibited two phases: the first occurring during the first 3-hour portion of the incubation was an inhibition, while the second was a promotive effect at about the 5th hour of incubation. Both of these effects were red, far redreversible, implicating phytochrome in this effect. Neither the destruction nor the immobilization of this exogenous indoleacetic acid apeared to be greatly affected by red light irradiation. The principal interaction between phytochrome and indoleacetic acid appears to occur during the absorption of exogenous indoleacetic acid. This effect may be related to the control by phytochrome of the amount of auxin which diffuses from coleoptile tips.     

Further support for the involvement of phytoehrome in stomatal movement

The involvement of phytochrome in stomatal movement in Commelina communis L. is indicated by the following observations: 1) Short irradiation with red or blue light causes opening, of isolated stomata and swelling of guard cell protoplasts. This is reversed by subsequent far red irradiation. 2) In a similar way, stomatal response to prolonged irradiation with red or blue light is decreased by concomitant far red irradiation. 3) Pretreatment with filipin, which interferes with phytochrome binding to membranes, decreases stomatal opening in red and blue light. The stomatal responses to blue and red light are modified by DCMU, N₂, CO_2-enriched atmosphere, and CO_2-free air, which are known to affect, among other processes, chlorophyll fluorescence. Increased chlorophyll fluorescence by DCMU, N₂ and CO₂-enriched atmosphere enhanced stomatal opening in blue light and inhibited it in red light. CO₂-free air, which decreases chlorophyll fluorescence, had the opposite effect.     

The active form of phytochrome: a new hypothesis based on phytochrome pelletability studies

Difficulties arising from the current dogma that the far-red absorbing form of phytochrome (P_fr) is the only active form are discussed.A new hypothesis is proposed in which phytochrome is held to be the photoreceptor for both low energy (pulse) and high energy (HIR) responses. There is a common basic mechanism of action involving interaction between phytochrome and a binding site within the cell. The phytochrome involvement in low energy responses exhibits an action spectrum for binding that matches the P_r absorption spectrum and reversibility by far-red irradiation. Upon prolonged irradiation the phytochrome-binding site interaction acquires different characteristics that are reminiscent of those displayed in HIR, e.g. dependence on sustained irradiation for continual binding, dependence of the degree of binding on irradiance and the similarity of the action spectrum with that of HIR action spectra, e.g. that for inhibition of lettuce hypocotyl lengthening.As expected on the basis of the new hypothesis the particulate fraction of phytochrome contains both P_r and P_fr. Arguments are advanced that the presence of P_r in pellets of particulate phytochrome cannot be accounted for by (i) the “induced fit” hypothesis, (ii) the “pigment cycling” hypothesis, and (iii) the “open phytochrome-receptor model”. We conclude that phytochrome molecules, after being sufficiently energized can interact with their intracellular binding sites irrespective of their chromophoric configuration.     

Studies on Greening of Etiolated Seedlings II. Leaf Greening by Phytochrome Action in the Embryonic Axis

We could demonstrate that greening of primary bean leaves in etiolated seedlings of Phaseolus vulgaris cv. Limburg can be controlled by a selective light-pretreatment of the embryonic axis. This light-induced interorgan synergism proved to be a phytochrome-mediated process. The red/farred photoreversible effect on the embryonic axis seems to be primarily linked to changes in the energy metabolism of the primary leaves. Phototransformation of the protochlorophyll present and pigment synthesis are very dependent upon an adequate supply of biochemical energy. When the embryonic axis is selectively pre-exposed to red light for a short time, respiration is markedly enhanced in the leaves and photosynthesis starts immediately upon illumination of the etiolated leaves after an incubation period of optimal length in the dark. The stimulatory effect of the red pretreatment on leaf respiration and photosynthetic capacity could be abolished to the level of the dark controls by a subsequent far-red irradiation on the embryonic axis. It is therefore postulated that phytochrome plays a regulatory role in interorgan cooperation. The metabolic changes involved in photomorphogenesis of etiolated seedlings are closely related to changes in energy production. Our data indicate that the primary act of phytochrome becomes operative at the biochemical level by its directional influence on the energy balance of the cell and coordinates the use of metabolic energy within a tissue and between organs.     

Phytochrome‐mediated regulation of plant respiration and photorespiration

The expression of genes encoding various enzymes participating in photosynthetic and respiratory metabolism is regulated by light via the phytochrome system. While many photosynthetic, photorespiratory and some respiratory enzymes, such as the rotenone‐insensitive NADH and NADPH dehydrogenases and the alternative oxidase, are stimulated by light, succinate dehydrogenase, subunits of the pyruvate dehydrogenase complex, cytochrome oxidase and fumarase are inhibited via the phytochrome mechanism. The effect of light, therefore, imposes limitations on the tricarboxylic acid cycle and on the mitochondrial electron transport coupled to ATP synthesis, while the non‐coupled pathways become activated. Phytochrome‐mediated regulation of gene expression also creates characteristic distribution patterns of photosynthetic, photorespiratory and respiratory enzymes across the leaf generating different populations of mitochondria, either enriched by glycine decarboxylase (in the upper part) or by succinate dehydrogenase (in the bottom part of the leaf).     

Phytochrome-membrane interactions as a factor in stomatal opening

Red light enhances stomatal opening in Commelina communis L. This light effect is reversed by far-red irradiation. Pretreatment with filipin, which competitively inhibits phytochrome binding to membranes, also inhibits light-enhanced opening. The pretreatment with filipin is more inhibitory if preceded by red irradiation, than after far-red irradiation. Similar results are obtained with cycloheximide and low temperature, which retard phytochrome synthesis more than its degradation. This result may indicate an enhanced release of phytochrome in the Pfr form from binding sites rather than release of phytochrome in the Pr form. This points towards the possibility that phytochrome degradation and its release from binding sites are coupled.     

Photocontrol of petiole elongation in light-grown strawberry plants

Summary The mode of phytochrome control of elongation growth was studied in fully-green strawberry (Fragaria x Ananassa Duch.) plants. Petiole growth showed two distinct types of response to light. In one, the end-of-day response, petioles were lengthened by low-intensity far-red irradiation for 1 h immediately following the 8 h photoperiod. The response was little or no greater with prolonged exposure and less when the start of far-red was delayed. It was already evident in the first leaf to emerge after treatment began. With the development of successive leaves a second, photoperiodic, type of response appeared, in which petioles lengthened following only prolonged exposure to red, far-red, mixtures of the two, or tungsten lighting, all at low levels of intensity. As with the inhibition of flowering in previous experiments, irradiation with red light during the second half of the otherwise long dark period gave the greatest response.Abbreviations and Symbols FR far-red light - HIR high irradiance response - R red light - P_r phytochrome in the red light absorbing form - P_fr phytochrome in the far-red light absorbing form - SDP short-day plant - LDP long-day plant - PAR photosynthetically active radiation     

Seed germination of Arabidopsis thaliana phyA/phyB double mutants is under phytochrome control.

We examined the photocontrol of seed germination in the phyA/phyB double mutants of Arabidopsis thaliana seeds. Dormant phyA/phyB seeds showed a red/far-red light (R/FR)-reversible induction of seed germination. This suggests the involvement of at least one other phytochrome, phyC, D, and/or E, in controlling seed germination. We designated this spectrally active phytochrome in phyA/phyB as phyX. The full reversibility of the R-induced germination by subsequent FR pulses, and the observation that the response is reversible by FR, even after a 3-h R treatment, indicates that this phyX response belongs to the low-fluence-response type. Thus, this phyX response is functionally related to phyB-mediated responses. However, in contrast to phyB-controlled seed germination, this phyX-mediated response needs a prolonged imbibition period and exhibits reversibility kinetics different from that needed for phyB. Furthermore, this phyX response requires a prolonged irradiation time and shows a fluence rate response dependency, showing a similarity to the high irradiance response of photomorphogenesis. Thus, phyX, with regard to its control of seed germination, is a functionally new phytochrome that shares some characteristics of both phyA- and phyB-mediated responses.