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     

Integral association of phytochrome with a membranous fraction fromAvena shoots: in vivo characterization and physiological significance

Phytochrome in the far-red light absorbing form (Pfr) was observed to disappear in vivo more rapidly from the non-cation-requiring pelletable phytochrome population than from the supernantant phytochrome population of oat seedlings given an increasing dark incubation after red irradiation. The amount of pelletable phytochrome in the red light absorbing form (Pr) remained relatively stable while supernatant Pr was lost. These observations indicated that supernant Pfr was subject to loss during the incubation, while pelletable Pfr was subject to both dark reversion and loss.During the incubation, the ability of far-red irradiation to reverse the red-induced increase in phytochrome pelletability was lost, with kinetics similar to those of the loss of pelletable Pfr.Far-red reversibility of the red-induced increase in coleoptile elongation correlated with the change intotal Pfr in both supernatant and pelletable phytochrome populations, but with the change in the ratio of Pfr to total phytochrome only in the pelletable phytochrome population.The possible significance of these results is discussed with reference to the action of phytochrome in the photocontrol of physiological growth responses.Abbreviations Pfr phytochrome in the far-red light absorbing form - Pr phytochrome in the red absorbing form - Ptot total phytochrome     

Intracellular localisation of phytochrome in oat coleoptiles by electron microscopy

The intracellular localisation of phytochrome in oat (Avena sativa L. cv. Garry Oat) coleoptiles was analysed by electron microscopy. Serial ultrathin sections of resin-embedded material were indirectly immunolabeled with polyclonal antibodies against phytochrome together with a gold-coupled second antibody. The limits of detectability of sequestered areas of phytochrome (SAPs) were analysed as a function of light pretreatments and amounts of the far-red absorbing form of phytochrome (Pfr) established. In 5-d-old dark-grownAvena coleoptiles SAPs were not detectable if less than 13 units of Pfr — compared with 100 units total phytochrome of 5-d-old dark-grown seedlings — were established by a red light pulse. In other sets of experiments, seedlings were preirradiated either with a non-saturating red light pulse to allow destruction to occur or with a saturating red followed by a far-red light pulse to induce first SAP formation and then its disaggregation. These preirradiations resulted in an increase of the limit of detectability of SAP formation after a second red light pulse to 38–41 and 19–23 units Pfr, respectively. We conclude that with respect to Pfr-induced SAP formation an adaptation process exists and that our data indicate that SAP formation is not a simple self-aggregation of newly formed Pfr.Abbreviations FR far-red light - Pfr, Pr far-red-absorbing and red-absorbing forms of phytochrome, respectively - Plot total phytochrome (Pfr + Pr) - R red light - SAP sequestered areas of phytochrome This work was supported by Deutsche Forschungsgemeinschaft (SFB 206). The competent technical assistance of Karin Fischer is gratefully acknowledged.     

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     

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     

The involvement of phytochrome in controlling chlorophyll and 5-aminolevulinate formation in a gymnosperm seedling (Pinus sylvestris)

Chlorophyll a (Chl a) accumulation in the cotyledons of Scots pine seedlings (Pinus sylvestris L.) is much higher in the light than in darkness where it ceases 6 days after germination. When these darkgrown seedlings are treated with continuous white light (3,500 lx) a 3 h lag phase appears before Chl a accumulation is resumed. The lag phase can be eliminated by pretreating the seedlings with 7 h of weak red light (0.14 Wm^-2) or with 14 red light pulses separated by relatively short dark periods (<100 min). The effect of 15s red light pulses can be fully reversed by 1 min far-red light pulses. This reversibility is lost within 2 min. In addition, the amount of Chl a formed within 27 h of continuous red light is considerably reduced by the simultaneous application of far-red (RG 9) light. It is concluded that phytochrome (P_fr) is required not only for the elimination of the lagphase but also to maintain a high rate of Chl a accumulation in continuous light. Since accumulation of 5-aminolevulinate (ALA) responds in the same manner as Chl a accumulation to a red light pretreatment it is further concluded that ALA formation is the point where phytochrome regulates Chl biosynthesis in continuous light. No correlation has been found between ALA and Chl a formation in darkness. This indicates that in a darkgrown pine seedling ALA formation is not rate limiting for Chl a accumulation.Abbreviations Chl chlorophyll(ide) - PChl protochlorophyll(ide) - ALA 5-aminolevulinate - P_r the red absorbing form of phytochrome - P_fr the far-red absorbing form of phytochrome - P_tot total phytochrome ([P_r]+[P_fr])     

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     

Reorientation of microtubules at the outer epidermal wall of maize coleoptiles by phytochrome,blue-light photoreceptor,and auxin

Summary The effects of red and blue light on the orientation of cortical microtubules (MTs) underneath the outer epidermal wall of maize (Zea mays L.) coleoptiles were investigated with immunofluorescent techniques. The epidermal cells of dark-grown coleoptiles demonstrated an irregular pattern of regions of parallel MTs with a random distribution of orientations. This pattern could be changed into a uniformly transverse MT alignment with respect to the long cell axis by 1 h of irradiation with red light. This response was transient as the MTs spontaneously shifted into a longitudinal orientation after 1–2 h of continued irradiation. Induction/reversion experiments with short red and far-red light pulses demonstrated the involvement of phytochrome in this response. In contrast to red light, irradiation with blue light induced a stable longitudinal MT alignment which was established within 10 min. The blue-light response could not be affected by subsequent irradiations with red or far-red light indicating the involvement of a separate blue-light photoreceptor which antagonizes the effect of phytochrome. In mixed light treatments with red and blue light, the blue-light photoreceptor always dominated over phytochrome which exhibited an apparently less stable influence on MT orientation. Long-term irradiations with red or blue light up to 6 h did not reveal any rhythmic changes of MT orientation that could be related to the rhythmicity of helicoidal cell-wall structure. Subapical segments isolated from dark-grown coleoptiles maintained a longitudinal MT arrangement even in red light indicating that the responsiveness to phytochrome was lost upon isolation. Conversely auxin induced a transverse MT arrangement in isolated segments even in blue light, indicating that the responsiveness to blue-light photoreceptor was eliminated by the hormone. These complex interactions are discussed in the context of current hypotheses on the functional significance of MT reorientations for cell development.Abbreviations MT cortical microtubule - P_r, P_fr red and far-red absorbing form of phytochrome     

Light conditions inside developing buds affect floral induction

Buds of Pinus sylvestris L. and Picea abies (L.) Karst. were pierced with optical fibres allowing natural light to the vicinity of apical domes. Induction of female strobiles was achieved. Since bud scales absorb more red than far-red light, the treatment may have increased the far-red light absorbing form of phytochrome.Abbreviations Pfr far-red light absorbing form of phytochrome     

Phytochrome-controlled extension growth of Avena sativa L. seedlings

The effects of continuous red and far-red light and of brief light pulses on the growth kinetics of the mesocotyl, coleoptile, and primary leaf of intact oat (Avena sativa L.) seedlings were investigated. Mesocotyl lengthening is strongly inhibited, even by very small amounts of P_fr, the far-red light absorbing form of phytochrome (e.g., by [P_fr]0.1% of total phytochrome, established by a 756-nm light pulse). Coleoptile growth is at first promoted by P_fr, but apparently inhibited later. This inhibition is correlated in time with the rupturing of the coleoptile tip by the primary leaf, the growth of which is also promoted by phytochrome. The growth responses of all three seedling organs are fully reversible by far-red light. The apparent lack of photoreversibility observed by some previous investigators of the mesocotyl inhibition can be explained by an extremely high sensitivity to P_fr. Experiments with different seedling parts failed to demonstrate any further obvious interorgan relationship in the light-mediated growth responses of the mesocotyl and coleoptile. The organspecific growth kinetics, don't appear to be influenced by P_fr destruction. Following an irradiation, the growth responses are quantitatively determined by the level of P_fr established at the onset of darkness rather than by the actual P_fr level present during the growth period.Abbreviation P_fr far-red light absorbing form of phytochrome     

A light-regulated pool of phytochrome and rudimentary high-irradiance responses under far-red light in Pinus elliottii and Pseudotsuga menziesii

The presence of a phytochrome pool down-regulated by light and the occurrence of high-irradiance responses to far-red light are well documented in angiosperms but not in gymnosperms. A pool of phytochrome was identified in Pinus elliottii and Pseudotsuga menziesii seedlings grown in darkness with a monoclonal antibody developed against oat phytochrome A. This pool was barely detectable in light-grown tissues. Dark-grown conifer seedlings transferred to continuous red light showed a gradual decrease of the levels of immunodetectable phytochrome. This decrease was significantly slower in gymnosperms than in angiosperms. Dark-grown seedlings of P. elliottii and P. menziesii showed enhanced growth of the cotyledonary whorl and increased anthocyanin pigmentation of the hypocotyl, but no hypocotyl-growth inhibition in response to continuous far-red light. Hourly pulses were significantly less effective than continuos far-red light. The response to far-red light was not observed in seedlings pretreated with red light to reduce the levels of immunodetectable phytochrome. Rudiments of phytochrome A-like function and kinetics are present in P. elliottii and P. menziesii.     

Phytochrome-mediated induction of phenylalanine ammonia-lyase in the cotyledons of tomato (Lycopersicon esculentum Mill.) plants

Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5.) induction in cotyledons from 96-h dark-grown Lycopersicon esculentum Mill. was studied in response to continuous light and hourly light pulses (blue, red, far red). The increases of PAL promoted by blue and red pulses are reversed completely by immediately following 758 nm irradiations. The response to continuous red light could be substituted for by hourly 6-min red light pulses. The effect of continuous red treatments is mainly due to a multiple induction effect of phytochrome. In contrast to red light, hourly light pulses with far red and blue, light can only partially substitute for continuous irradiation. The continuous blue response could be due to a combination of a multiple induction response and of a high irradiance response of phytochrome. The continuous far red response, could represent a high irradiance response of phytochrome. Dichromatic irradiations indicate that phytochrome is the photoreceptor controlling the light response (PAL) in tomato seedlings.Abbreviations Norflurazon NF-4-chloro-5-(methylamino)-2-(,,,-trifluoro-m-tolyl)-3 (2H) pyridazinone - PAL phenylalanine ammonia-lyase - phytochrome photoequilibrium P_fr/P_tot - P_fr far-red absorbing form of phytochrome - P_r red absorbing form of phytochrome - P_tot total phytochrome: P_r+P_fr     

Light-induced changes in the photoresponses of plant stems the loss of a high irradiance response to far-red light

De-etiolation results in phytochrome destruction, greening, and the loss of the far-red high irradiance responses (HIR). Evidence is presented against the hypothesis that the loss of the far-red HIR is a direct consequence of phytochrome destruction. Loss of the far-red HIR for the inhibition of elongation in hypocotyls of Raphanus sativus involves two different, but linked, actions of phytochrome. An induction reaction requires the far-red absorbing form of phytochrome for about 20 min after which accumulation of its product depends only on time. A second reaction requires continuous light or frequent short irradiations and involves cycling of the phytochrome system. This acts on the product of the induction reaction. It is proposed that in green plants an important mode of operation of phytochrome in the light depends on pigment cycling, and that during de-etiolation this system is established under phytochrome control.Abbreviations HIR high irradiance response - R red - FR farred light - P_tot phytochrome, P_r its red absorbing form, P_fr its far-red absorbing form A.M. Jose was the holder of Ministry of Agriculture, Fisheries and Food award AE 6819     

The light-induced reduction of the gravitropic growth-orientation of seedlings of Arabidopsis thaliana (L.) Heynh. is a photomorphogenic response mediated synergistically by the far-red-absorbing forms of phytochromes A and B

Hypocotyls of dark-grown seedlings of Ara bidosis thaliana exhibit a strong negative gravitropism, which is reduced by red and also by long-wavelength, far-red light treatments. Light treatments using phytochrome A (phyA)- and phytochrome B (phyB)-deficient mutants showed that this response is controlled by phyB in a red/far-red reversible way, and by phyA in a non-reversible, very-low-fluence response. Crosses of the previously analyzed phyB-1 allele (in the ecotype Landsberg erecta background) to the ecotype Nossen wild-type (WT) background resulted in a WT-like negative gravitropism in darkness, indicating that the previously described gravitropic randomization observed with phyB-1 in the dark is likely due to a second mutation independent of that in the PHYB gene.Abbreviations FR long-wavelength far-red light - phyA phytochrome A (holoprotein) - phyB phytochrome B (holoprotein) - Pr red-absorbing form of phytochrome - WT wild type We thank Dr. A. Nagatani (RIKEN Institute, Wako-City, Japan) and Dr. M. Furuya (Hitachi, Hatoyama, Japan) for the phyA-201/phyB-5 double mutant. The work was supported by Deutsche Forschungsgemeinschaft and Human Frontier Science Program grants to E.S.     

Action spectra for changes in the “high irradiance reaction” in hypocotyls of Sinapis alba L.

Detailed action spectra are presented for the inhibition of hypocotyl extension in dark-grown Sinapis alba L. seedlings by continuous (24 h) narrow waveband monochromatic light between 336 nm and 783 nm. The results show four distinct wavebands of major inhibitory action; these are centred in the ultra-violet (_max=367 nm), blue (_max=446 nm), red (_max=653 nm) and far-red (_max=712 nm) wavebands. Previous irradiation of the plants with red light (which also decreases Ptot) causes decreased inhibitory action by all wavelengths except those responsible for the red light inhibitory response. Pre-irradiation did not alter the wavelength of the action maxima. It is concluded that ultra-violet and blue light act mainly on a photoreceptor which is different from phytochrome.Abbreviations B blue - D dark - FR far-red - HIR high irradiance reaction - HW half power bandwith - Pr R absorbing form of phytochrome - Pfr FR absorbing form of phytochrome - Ptot total phytochrome=Pr+Pfr - R red - UV ultra violet     

Action spectrum and characteristics of the light activated disappearance of phytochrome in oat seedlings

The action spectrum for the light-activated destruction of phytochrome in etiolated Avena seedlings has been determined. There are 2 broad maxima, one between 380 and 440 mμ, the other between 600 and 700 mμ. peaking at about 660 mμ. On an incident energy basis, the red region of the spectrum is more efficient than the blue by about one order of magnitude in activating phytochrome disappearance. Both the red absorbing as well as the far-red absorbing forms of phytochrome are destroyed after exposure of Avena seedling to either red or blue light.

From the action spectrum and photoreversibility of pigment loss, we conclude that phytochrome acts as a photoreceptor for the photoactivation of its metabolically-based destruction. We suggest that another pigment might also be associated with the disappearance of phytochrome in oat seedlings exposed to blue light.

     

Control by light of hypocotyl growth in de-etiolated mustard seedlings

After sowing, mustard (Sinapis alba L.) seedlings were grown for 48 h in white light (25°C). These fully de-etiolated, green seedlings were used as experimental material between 48 and 72 (84) h after sowing. The question researched was to what extent control by light of hypocotyl elongation is due to phytochrome in these seedlings. It was found that the light effect on hypocotyl growth is very probably exerted through phytochrome only. In particular, we found no indication for the involvement of a specific blue light photoreceptor pigment.Abbreviations HIR high irradiance reaction - P_fr far-red absorbing, physiologically active form of phytochrome - P_r red absorbing, physiologically inactive form of phytochrome - Pot total phytochrome, i.e. [P_r]+[P_fr] - [P_fr]/[P_tot] - red red light - fr far-red light - wl white light - bl blue light - di dichromatic irradiation - l hypocotyl length     

Intracellular redistribution of phytochrome in etiolated soybean (Glycine max L.) seedlings

The intracellular distribution of phytochrome in hypocotyl hooks of etiolated soybean (Glycine max L.) has been examined by immunofluorescence using a newly produced monoclonal antibody (Soy-1) directed to phytochrome purified from etiolated soybean shoots. Cortical cells in the hook region exhibit the strongest phytochrome-associated fluorescence, which is diffusely distributed throughout the cytosol in unirradiated, etiolated seedlings. A redistribution of immunocytochemically detectable hytochrome to discrete areas (sequestering) following irradiation with red light requires a few minutes at room temperature in soybean, whereas this redistribution is reversed rapidly following irradiation with far-red light. In contrast, sequestering in oat (Avena sativa L.) occurs within a few seconds (D. McCurdy and L. Pratt, 1986, Planta 167, 330–336) while its reversal by far-red light requires hours (J. M. Mackenzie Jr. et al., 1975, Proc. Natl. Acad. Sci. USA 72, 799–803). The time courses, however, of red-light-enhanced phytochrome pelletability and sequestering are similar for soybean as they are for oat. Thus, while these observations made with a dicotyledon are consistent with the previous conclusion derived from work with oat, namely that sequestering and enhanced pelletability are different manifestations of the same intracellular event, they are inconsistent with the hypothesis that either is a primary step in the mode of action of phytochrome.Abbreviations DIC differential interference contrast - FR far-red light - Ig immunoglobulin - Pfr, P far-red- and red-absorbing form of phytochrome, respectively - R red light This work was supported by National Science Foundation grant No. DCB-8703057.     

Light quantity and quality interactions in the control of elongation growth in light-grown Chenopodium rubrum L. seedlings

The spectral control of hypocotyl elongation in light-grown Chenopodium rubrum L. seedlings has been studied. The results showed that although the seedlings responded to changes in the quantity of combined red and far-red radiation, they were also very sensitive to changes in the quantity of blue radiation reaching the plant. Altering the proportion of red: far-red radiation in broad waveband white light caused marked differences in hypocotyl extension. Comparison of the responses of green and chlorophyll-free seedlings indicated no qualitative difference in the response to any of the light sources used, although photosynthetically incompetent plants were more sensitive to all wavelengths. Blue light was found to act primarily of a photoreceptor which is different from phytochrome. It is concluded that hypocotyl extension rate in vegetation shade is photoregulated by the quantity of blue light and the proportion of red: far-red radiation. In neutral shade, such as that caused by stones or overlying soil, hypocotyl extension appears to be regulated primarily by the quantity of light in the blue waveband and secondarily by the quantity of light in the red and far-red wavebands.Abbreviations B blue - FR far-red - k ₁, k ₂ rate constants for photoconverison of Pr to Pfr and Pfr to Pr, respective - k ₁/k ₁ +k ₂= phytochrome photoequilibrium - k ₁ +k ₂= phytochrome cycling rate - Pr=R absorbing form of phytochrome - Pfr=FR absorbing form of phytochrome - P_tot Pr+Pfr - PAR photosynthetically active radiation = 400–700 nm - R red - WL white light