Photomanipulation of phytochrome in lettuce seeds

Seeds of lettuce (Lactuca sativa L. cv. Grand Rapids) were imbibed and given either short irradiation with red or far red light prior to drying or dried under continuous red or far red light. Seeds treated with either short or continuous red germinate in darkness, whereas seeds treated with either short or continuous far red require a short exposure to red light, after a period of imbibition, to stimulate germination. Irradiation of dry red seeds with far red light immediately before sowing results in a marked inhibition of germination. This result was predicted since far red-absorbing form phytochrome can be photoconverted to the intermediate P650 (absorbance maximum 650 nm) in freeze-dried tissue. A similar far red treatment to continuous red seeds is less effective and it is concluded that in these seeds a proportion of total phytochrome is blocked as intermediates between red-absorbing and far red-absorbing form phytochrome, which only form the far red-absorbing form of phytochrome on imbibition. The inhibition of dry short red seeds by far red light can be reversed by an irradiation with short red light given immediately before sowing, confirming that P650 can be photoconverted back to the far red-absorbing form of phytochrome. The results are discussed in relation to seed maturation (dehydration) on the parent plant.     

In vivo phytochrome reversion in immature tissue of the alaska pea seedling

Reversion of far red-absorbing phytochrome to red-absorbing phytochrome without phytochrome destruction (that is, without loss of absorbancy and photoreversibility) occurs in the following tissues of etiolated Alaska pea seedlings (Pisum sativum L.): young radicles (24 hours after start of imbibition), young epicotyls (48 hours after start of imbibition), and the juvenile region of the epicotyl immediately subjacent to the plumule in older epicotyls. Reversion occurs rapidly in the dark during the first 30 minutes following initial phototransformation of red-absorbing phytochrome to far red-absorbing phytochrome. If these tissues are illuminated continuously with red light for 30 minutes, the total amount of phytochrome remains unchanged. Beyond 30 minutes after a single phototransformation or after the start of continuous red irradiation, phytochrome destruction commences. In young radicles, sodium azide inhibits this destruction, but does not affect reversion. In older tissues in which far red-absorbing phytochrome destruction begins immediately upon phototransformation, strong evidence for simultaneous far red-absorbing phytochrome reversion is obtained from comparison of far red-absorbing phytochrome loss in the dark following a single phototransformation with far red-absorbing phytochrome loss under continuous red light.     

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.     

Red and far red effects on phenylalanine ammonia-lyase in raphanus and sinapis seedlings do not correlate with phytochrome spectrophotometry

In seedlings of Raphanus sativus (radish) and Sinapis alba (mustard), irradiation for 6 hours with far red light significantly increases the extractable activity of phenylalanine ammonia-lyase by the end of the light period. A schedule of 10 minutes red light-110 minutes darkness-10 minutes red-110 minutes darkness-10 minutes red-110 minutes darkness has no effect as compared to dark controls. However, the red light program maintains a level of far red-absorbing phytochrome always measurable by in vivo spectrophotometry during the 6-hour experimental period. We conclude that the far red effect on this enzyme and for this specific material cannot be explained solely by formation and maintenance of far red-absorbing phytochrome.     

Phytochrome properties and the molecular environment

In vitro data support a scheme of phytochrome phototransformation involving intermediates in a sequential pathway. The fraction of total phytochrome maintained as intermediate under conditions of pigment cycling as well as the rate of the dark reversion of the far red-absorbing (Pfr) to the red-absorbing form of phytochrome (Pr) has been shown to depend on the molecular environment of the phytochrome molecules. Inverse dark reversion of Pr to Pfr has been observed in vitro. These results contribute toward an understanding of the observed paradoxes between physiological experiments and measurements of the amount and state of phytochrome in vivo. The in vivo spectrophotometric assay measures an average of the properties of phytochrome in different cellular environments, whereas a particular physiological response may be controlled by phytochrome molecules in one particular environment. It is therefore possible that all phytochrome is potentially active and triggers specific responses by virtue of its localization.     

Inhibition of Prechill-induced Dark Germination in Sorghum halepense (L.) Pers. Seeds by Phytochrome Transformations

A 10 C dark prechilling of johnsongrass [Sorghum halepense (L.) Pers.] seeds, when terminated by a 2-hr, 40 C temperature shift, potentiates about 40% germination at 20 C in darkness. Irradiation of the seeds before, during, and at the end of prechilling with far red light reduces the subsequent germination, although red irradiation after the far red can overcome some of the inhibition. However, either brief red or far red irradiation given immediately after the temperature shift inhibits subsequent germination by one-third to one-half. The results suggest that the far red-absorbing form of phytochrome is a factor in the prechill-induced dark germination and that phytochrome participates in the inhibition of germination by irradiations immediately after the temperature shift.     

Changes in Phytochrome Expressed by Germination of Amaranthus retroflexus L. Seeds

Effects of red (600 to 680 nanometers) and far red (700 to 760 nanometers) irradiances on Amaranthus retroflexus L. seeds indicate that synthesis of phytochrome in the red-absorbing form takes place in water-imbibed nongerminating seeds at 35 C. After 96 hours in darkness, conversion of about 0.10% phytochrome to the far red-absorbing form induces 50% germination. Continuous far red radiation at 35 C with an irradiance of 0.4 × 10⁻¹⁰ Einsteins per square centimeter per second caused photoinactivation of phytochrome about equal to the rate of synthesis. Germination of seeds at 35 C, following far red irradiation adequate to establish the photostationary state, is enhanced by holding at 26 C for 16 minutes. Germination is unaffected relative to controls at constant temperature, if the period at 26 C precedes irradiation. The results indicate a quick response to action of phytochrome in a germination process.     

Characterization of the Destruction of Phytochrome in the Red-absorbing Form

Both the red-absorbing (Pr) and far red-absorbing (Pfr) forms of phytochrome undergo destruction, defined as the loss of photoreversibly detectable chromoprotein following actinic irradiation of dark-grown tissue, in 4-day-old etiolated oat seedlings. Pr and Pfr destruction follow the same time course, exhibit the same time delay after actinic irradiation when the plants are grown in sealed containers, result in a loss of antigenically detectable phytochrome, as determined by radial immunodiffusion assay, equal to the loss of spectrophotometrically detectable phytochrome, and have the same sensitivity to 2-mercaptoethanol and azide. We suggest that Pr destruction is a consequence of the same mechanism that is responsible for Pfr destruction.     

Intracellular localisation of phytochrome and ubiquitin in red-light-irradiated oat coleoptiles by electron microscopy

The intracellular localisation of phytochrome and ubiquitin in irradiated oat coleoptiles was analysed by electron microscopy. We applied indirect immunolabeling with polyclonal antibodies against phytochrome from etiolated oat seedlings or polyclonal antibodies against ubiquitin from rabbit reticulocytes, together with a goldcoupled second antibody, on serial ultrathin sections of resin-embedded material. Immediately after a 5-min pulse of red light-converting phytochrome from the red-absorbing (Pr) to the far-redabsorbing (Pfr) form-the label for phytochrome was found to be sequestered in electron-dense areas. For up to 2 h after irradiation, the size of these areas increased with increasing dark periods. The ubiquitin label was found in the same electrondense areas only after a dark period of 30 min. A 5 min pulse of far-red light, which reverts Pfr to Pr, given immediately after the red light did not cause the electron-dense structures to disappear; moreover, they contained the phytochrome label immediately after the far-red pulse. In contrast, after the reverting far-red light pulse, ubiquitin could only be visualised in the electron-dense areas after prolonged dark periods (i.e. 60 min). The relevance of these data to light-induced phytochrome pelletability and to the destruction of both Pr and Pfr is discussed.Abbreviations FR far-red light; Pfr - Pr far-red-absorbing and red-absorbing forms of phytochrome, respectively - R red light     

Phytochrome — all regions marked by a set of monoclonal antibodies reflect conformational changes

Monoclonal antibodies to defined locations on six regions of the phytochrome molecule (from Avena sativa L. or Zea mays L.) were each found to have a different affinity toward the farred-absorbing form of phytochrome (Pfr) and the red-absorbing form (Pr). The differences were small, but were consistently shown by antibodies which bind to the vicinity of the aminoterminus, the carboxylterminus and to sequences in between. It seems that the conformational differences between Pr and Pfr extend over the whole molecule in as far as it is represented by these regions and the antibodies binding to them.Abbreviations Pfr far-red-absorbing form of phytochrome - Pr red-absorbing form of phytochrome     

In Vivo Properties of Membrane-bound Phytochrome

After a 3-minute irradiation with red light, which saturates the phototransformation from the red light-absorbing form of phytochrome to the far red light absorbing form of phytochrome, about 40% of the phytochrome extractable from hooks of etiolated squash seedlings (Cucurbita pepo L. cv. Black Beauty) can be pelleted as Pfr at 17,000g after 30 minutes. Dark controls yield only 2 to 4% pelletable phytochrome in the form Pr. If a dark period intervenes between red irradiation and extraction, the bound Pfr gradually loses its photoreversibility. The time course for this destruction parallels the time course for phytochrome destruction in vivo following saturating red irradiation. The soluble fraction of phytochrome remains constant. These results suggest that in squash seedlings phytochrome destruction is related exclusively to the fraction which becomes membrane-bound. The induction of phytochrome binding by red light is not completely reversible by far red. In plants given saturating red followed immediately by saturating far red light, 12% of the phytochrome is found in the bound fraction as Pr if the phytochrome extraction is immediate. If a dark period intervenes between red-far red treatment and extraction, the bound phytochrome is released within 2 hours. A model of the binding properties of phytochrome, based on molecular interaction at the membrane is proposed, and possible consequences for the mechanism of action of phytochrome are discussed.     

Polyclonal antibodies raised to phycocyanins contain components specific for the red-absorbing form of phytochrome

Polyclonal antibodies raised in rabbits to a mixture of sodium-dodecyl-sulphate-denatured C- and allo-phycocyanin, isolated from Anabaena cylindrica, cross-react with 124-kilodalton (kDa) phytochrome from etiolated oats, in enzyme-linked immunosorbent assays and on Western blots. The component(s) of the anti-phycocyanin serum that cross-reacts with phytochrome appears to be specific for the red-absorbing form of phytochrome (Pr). These antibodies can be detached from Pr by irradiation with red light, and thus show photoreversible binding. This property has been used to immunopurify the anti-phytochrome component from the antiserum using red light as the eluting agent. Competition assays and epitope-mapping studies indicate that the anti-phytochrome component may bind to a site located between 6 and 10 kDa from the amino-terminus of etiolated oat phytochrome.Abbreviations ELISA enzyme-linked immunosorbent assay - kDa kilodaton - FR far-red light - Pfr far-red-light-absorbing form of phytochrome - Pr red-light-absorbing form of phytochrome - R red light - SDS sodium dodecyl sulphate     

Turnover of phytochrome in pumpkin cotyledons

By using density labeling, it was found that the protein moiety of phytochrome is synthesized de novo in the red-absorbing form in cotyledons of dark-grown pumpkin (Cucurbita pepo L.) seedlings, as well as those irradiated with red light and returned to the dark. The rate of synthesis appears to be unaffected by the light treatment. Turnover of the red-absorbing form was also detected in dark grown seedlings using density labeling, while turnover of the far red-absorbing form is already implied from the well known “destruction” observed in irradiated seedlings. In both cases, true degradation of the protein is involved, but the rate constant of degradation of the far red-absorbing form may be up to two orders of magnitude greater than that of the red-absorbing form. The data indicate that, in pumpkin cotyledons, phytochrome levels are regulated against a background of continuous synthesis through divergent rate constants of degradation of the red and far red-absorbing forms and the relative proportions of the two forms present.     

Red light-induced accumulation of ubiquitin-phytochrome conjugates in both monocots and dicots

Phytochrome is rapidly degraded in vivo after photoconversion from the stable red-absorbing (Pr) form to the far red-absorbing (Pfr) form. Previously, we have shown in etiolated oat seedlings that ubiquitin-phytochrome conjugates (Ub-P) appear after Pfr formation suggesting that oat phytochrome is rapidly degraded by a ubiquitin-dependent proteolytic pathway. Here, we extend this observation to etiolated tissue from other monocotyledonous (corn [Zea mays. (L.)] and rye [Secale cereale (L.)] and dicotyledonous species (pea [Pisum sativum (L,)] and zucchini squash [Cucurbita pepo (L.)]). Following Pfr formation by red light, all four species synthesized a heterogeneous series of Ub-P that appeared and disappeared concomitant with the degradation of the chromoprotein. When Pfr was photoconverted back to Pr by a far-red light pulse, degradation of phytochrome ceased and the levels of Ub-P concomitantly dropped. In pea and zucchini squash, loss of Ub-P after photoconversion of Pfr back to Pr was rapid, occurring with a half-life of approximately 5 to 10 minutes. These data indicate that the accumulation of Ub-P after Pfr formation is a general phenomenon in etiolated seedlings of higher plants and further support the hypothesis that plants degrade Pfr via Ub-P intermediates.     

Discrimination between the red- and far-red-absorbing forms of phytochrome from Avena sativa L. by monoclonal antibodies

A set of rat monoclonal antibodies (ARC MAC 48 to 52 and 54 to 56), raised to phytochrome from dark-grown seedlings of Avena sativa L. was tested for the ability to discriminate between the red-absorbing (Pr) and far-red-absorbing (Pfr) forms of phytochrome by indirect enzyme-linked immunosorbent assay. MAC 50 bound more strongly to Pfr and MAC 49 and 52 showed preferential binding to Pr from extracts of dark-grown Avena seedlings; MAC 50 also bound more strongly to Pfr from brushite-purified phytochrome. The remainder of the monoclonal antibodies and a rabbit polyclonal antiphytochrome preparation did not discriminate between Pr and Pfr. The results provide evidence for conformational changes in defined regions of the phytochrome apoprotein upon photoconversion.Abbreviations ELISA enzyme-linked immunosorbent assay - FR far-red light - McAb monoclonal antibody(ies) - PBS phosphate-buffered saline - Pfr far-red-absorbing form of phytochrome - Pr red-absorbing form of phytochrome - R red light - PMSF phenylmethylsulphonylfluoride     

Phytochrome Control of Germination of Rumex crispus L. Seeds Induced by Temperature Shifts

High germination of curly dock (Rumex crispus L.) seeds is evident after suitable imbibition and temperature shift treatment, but germination at constant temperatures fails without an input of far red-absorbing form of phytochrome. Preliminary imbibitions at high temperatures (30 C) sharply reduce germination induced by temperature shifts. High germination may be restored by low energies of red radiation, or by brief far red adequate for the photosteady state. Prolonged far red during imbibition also nullifies temperature shift-induced germination. After prolonged far red, high germination may be restored by red radiation of an energy dependent upon the duration of the far red treatment. The evidence supports the conclusion that dark germination induced by temperature shifts arises from the interaction of pre-existent far red-absorbing form of phytochrome in the mature seeds with the temperature shift.     

Phytochrome photoreversibility: Empirical test of the hypothesis that it varies as a consequence of pigment compartmentation

Phytochrome of oat (Avena sativa L., cv. Garry) coleoptile cells in the red-light-absorbing form, Pr, is diffusely distributed while after conversion to the far-red-light-absorbing form, Pfr, it is observed only in very small areas within the cell. Comparison of phytochrome photoversibility measurements to the distribution of the pigment within the cell indicates that the spectral assay is not influenced by the observed compartmentalization of the chromoprotein. However, the observed compartmentalization of phytochrome is correlated with a loss in spectrophotometrically detectable Pr.Abbreviations Pr red-absorbing form of phytochrome - Pfr farred-absorbing form of phytochrome - R red light - FR far-red light C.I.W.-D.P.B. Publication No. 622     

Kinetics of the dichroic reorientation of phytochrome during photoconversion inMougeotia

In the green algaMougeotia, the dichroic orientation of the red-absorbing form of phytochrome (P_r) is parallel of the cell surface, whereas the far-red-absorbing form (P_fr) is oriented normal to it. The time course of the change from parallel to normal was investigated by double-flash irradiation with polarized red and far-red light. The results obtained by two different methods indicate that most of the phytochrome intermediates existing in the first 5 ms after the inducing red flash are still oriented parallel to the cell surface, similar to P_r. At increasing intervals between the red and the far-red flashes, more and more phytochrome molecules turn their transition moments to the P_fr orientation. This reaction is finished after approximately 30 ms. We conclude that the change in dichroic orientation of the phytochrome molecules inMougeotia occurs during the last relaxation steps of the intermediates on the way from P_r to P_fr. It cannot be decided yet, whether the first surface-normal phytochrome species is an intermediate or P_fr itself.Abbreviations P_r red-absorbing form of phytochrome - P_fr far-red-absorbing form of phytochrome A preliminary report of this work was presented at the European Symposium on Photomorphogenesis, University of Reading, UK (Kraml et al. 1982)     

Phytochrome Transformation and Action in Seeds of Rumex crispus L. during Secondary Dormancy

Promotion of germination by red light fails after prolonged dark imbibition of Rumex crispus L. seeds, indicative of a secondary dormancy. The degree and rate of inception of the dormancy increases with increasing temperature. Following establishment of the dormancy, germination response to red light can be restored by either prolonged cold treatment or brief high temperature shifts. Loss of phytochrome was not a factor in the initial establishment of the dormancy. When the seeds are in secondary dormancy, the chromophore of phytochrome can be transformed to the far red-absorbing form, but the far red-absorbing form cannot induce germination. The responses to changes in temperature suggested dependence of germination on order disorder transitions in components of the seeds.     

Differences in the physical properties of native and partially degraded phytochrome as probed by their differential sensitivity to permanganate oxidation

The differential sensitivities to permanganate oxidation of the red and far-red forms of native phytochrome from Avena sativa L. cv Mulaga (isolated as Pfr from red-irradiated tissue) and of partially degraded phytochrome (isolated as Pr from nonirradiated tissue) were determined. The far-red absorbing form of partially degraded phytochrome was 5 times more sensitive than its red-absorbing form, while both the far-red and red forms of native phytochrome exhibited identical sensitivity. The present data obtained with partially degraded phytochrome are in apparent agreement with the data and model of Hahn, Kang, and Song (1980 Biochem Biophys Res Commun 97: 1317-1323). Their model suggests that the chromophore of the red-absorbing form of phytochrome is buried in a hydrophobic crevice in the protein, while that of the far-red form is exposed. The data obtained with native phytochrome, however, are at variance with their model. Our data obtained with native phytochrome suggests that the chromophore of the red and the far-red absorbing forms of native phytochrome both are in a relatively protected environment and that only following partial proteolytic degradation of the phytochrome does the chromophore of its far-red form become relatively more exposed. The protective influence of the labile peptide could either be direct, because of its close physical proximity to the chromophore, or indirect, resulting in an alteration in chromophore-protein interaction.