Phytochrome intermediates and action spectra for light perception by dry seeds

It has previously been demonstrated that far-red irradiation of dry Lactuca sativa L. seeds results in inhibition of subsequent germination. Although red has no effect on dry seeds, a red irradiation following a farred irradiation reverses the effect of far-red. This phenomenon is most noticeable in seeds with artificially raised levels of phytochrome in the far-red absorbing form. Qualitatively similar results have been found for the seeds of Plantago major L., Sinapis arvensis L., and Bromus sterilis L. Action spectra studies on Plantago seeds show that the action peaks for promotion and inhibition of germination of hydrated seeds are at 660 and 730 nanometers, respectively. The action spectrum for inhibition of subsequent germination following irradiation of dry seeds is qualitatively and quantitatively similar to that for hydrated seeds, with an action peak at 730 nanometers, indicating absorption by phytochrome in the far-red absorbing form. However, the action spectrum for the reversal of this far-red effect on dry seeds has a broad peak at 680 nanometers and subsidiary peaks at 650 and 600 nanometers. It is proposed that this effect is due to light absorption by the phytochrome intermediate complex meta-Fa, and that the action spectrum reflects the in vivo absorption properties of this intermediate.     

The influence of season on adenine nucleotide concentrations and energy charge in four marsh plant species

Unilateral pulse ultraviolet irradiation caused positively phototropic coiling (> 180°) and curvature (≤ 180°) in the growth zone of dark-grown broom sorghum ( Sorghum bicolor Moench, cv. Acme Broomcorn and Sekishokuzairai Fukuyama) first internodes. Coiling was induced by irradiation at 257 to 302 nm, and proceeded to develop almost linearly during 72 h or more involving new tissue produced after irradiation. Curvature, caused at 308 to 413 nm and by red light, developed rapidly during the first several hours then slowly to cease by about 24 h, and did not surpass 120° even at the optimum photon fluences. Action spectra were higher towards shorter wavelengths, having a shoulder at about 287 nm, and could not separate the two photoresponses. The curvature was partially but markedly suppressed by far-red following the ultraviolet irradiation, whereas the coiling was not affected. Possible involvement of a specific UV-B photoreceptor and phytochrome in curvature and of a UV-C photoreceptor in coiling is discussed.     

Red Light-inhibited Mesocotyl Elongation in Maize Seedlings: II. Kinetic and Spectral Studies

Red light induces two distinct inhibition responses in mesocotyls of etiolated corn seedlings. A light dose of 10 nanoeinsteins per square centimeter is saturating for the more sensitive response, whereas doses above 1,000 nanoeinsteins per square centimeter are required to exceed the threshold sensitivity of the less sensitive one. The sensitive response can be detected within 20 minutes of the onset of illumination whereas the other response does not become apparent until more than 4 hours after the beginning of irradiation. The reciprocity law is valid for the first response, but probably not for the second. An action spectrum for the first response shows two maxima, one at 640 nanometers and the other between 660 and 670 nanometers, with a pronounced minimum near 650 nanometers. The effects both of 640 and 665 nanometers of light were reversible by far red light, but doses of far red required for full reversibility were almost three orders of magnitude greater than the doses of red required either to saturate the initial inhibition or to reverse the effect of far red light. The results suggest that corn may contain a red-absorbing pigment other than phytochrome which in some way interacts with phytochrome in the inhibition of mesocotyl elongation by red light.     

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.     

Action spectra for the inhibition of growth in radish hypocotyls

In etiolated seedlings of Raphanus sativus L. the inhibition of hypocotyl elongation by continuous light showed a major bimodal peak of action in the red and far-red, and two minor peaks in the blue regions of the spectrum. It is argued that, under conditions of prolonged irradiation, phytochrome is the pigment controlling the inhibition of hypocotyl elongation by red and far-red light, but that its mode of action in far-red is different from that in red. A distinct pigment is postulated for blue light.Abbreviations B blue - FR far red - G green - R red - HIR high irradiance reaction - P_r and P_fr red and far red absorbing forms of phytochrome - R red     

Relationship between Chloroplast Development and ent-Kaurene Biosynthesis in Peas

Treatment of etiolated pea (Pisum sativum (L. cv. Alaska) seedlings with 2′-isopropyl-4′-(trimethylammonium chloride)-5′-methylphenyl piperidine-1-carboxylate (Amo-1618) prior to irradiation with white light inhibits photomorphogenesis and formation and stacking of thylakoid membranes in the chloroplasts, as well as (−)-kaur-16-ene (ent-kaurene)biosynthesis. Exogenous gibberellic acid also inhibits greening. A crudely determined action spectrum for the photoinduction of ent-kaurene biosynthesis shows two peaks, one in the blue region at 458 to 490 nanometers and another in the red region at 606 to 678 nanometers. The possible participation of phytochrome in the photoinduction of ent-kaurene biosynthesis is indicated by comparative effects of red, far red, and alternating red/far red irradiations on enhancement of enzyme activity. The activity of blue light as well as red shows a similarity of the photoinduction of ent-kaurene synthesis activity to the high irradiance responses, and indicates probable participation of a second photoreceptor. From these observations, it is concluded that photoinduction of ent-kaurene biosynthesis and chloroplast development in shoots are closely linked processes.     

Action Spectrum of the Photoinduced Sexual Stage in the Fungus Nectria haematococca Berk. and Br. var cucurbitae (Snyder and Hansen) Dingley

An action spectrum was determined for the photoinduced formation of perithecia in a homothallic strain of Nectria haematococca Berk. and Br. var. cucurbitae (Snyder and Hansen) Dingley. Dose-response curves for perithecial formation were obtained from 340 to 510 nanometers at 10-nanometer intervals. Radiation longer than 510 nanometers was not effective for inducing perithecial formation. The action spectrum indicated peaks of activity near 360, 440, and 480 nanometers with shoulders near 420 and 460 nanometers. Minima occurred near 350 nanometers, 390 to 410 nanometers, and 470 nanometers. The general shape of this action spectrum appears to be similar to those obtained for many different near ultraviolet-blue-sensitive organisms in which a flavoprotein molecule was postulated to be the photoreceptor.     

Action Spectra for Chromatic Adaptation in Tolypothrix tenuis

The dark synthesis of biliproteins in the blue-green alga Tolypothrix tenuis is controlled by brief light treatments. Green light potentiates synthesis of phycoerythrin and red light potentiates synthesis of phycocyanin. Red reverses the effect of green and vice versa. Action spectra for the red and green effects were obtained for the wavelength region 320 nanometers to 710 nanometers, at 10-nanometer intervals. The principal action band in the red peaks at 660 nanometers, with a half-band width of 58 nanometers and an accompanying shortwave band at 360 nanometers. The green action band peaks at 550 nanometers, with a half-band width of 76 nanometers, and a shortwave band at 350 nanometers. Chromatic adaptation and another photomorphogenic response in the blue-green algae are discussed in terms of possible regulation by a photoreversible pigment recently isolated from Tolypothrix.     

The photosynthetic action spectrum of the bean plant

The photosynthetic action spectrum of the bean plant leaf, Phaseolus vulgaris L. (variety Red Kidney), has been determined with a diffraction grating illuminated by a 6500-watt xenon arc. An infrared CO₂ analyzer was used to determine the gross photosynthetic rate of the terminal leaflet of the first trifoliate leaf. The rate was measured as a function of the light intensity at steps of 12.5 nanometers which approximates the length of the leaflet used. Twenty-five curves between 400 and 700 nanometers were used to establish the action spectrum. All light curves were some linear function of the incident intensity, and all were extrapolated to zero. The action spectrum shows the following features. (a) there are two peaks (i.e., at about 670 and 630 nanometers) and a shoulder between 600 and 612 nanometers in the red region where the highest rate of photosynthesis is found. Lower peaks in descending order are found in the blue (at about 437 nanometers) and the green (at about 500 nanometers) regions. (b) There are two small minima at about 650 nanometers and between 470 and 480 nanometers, and a broad minimum is found between 540 and 530 nanometers. (c) The photosynthetic rate declines rapidly above 680 nanometers, reaching the lowest value at 700 nanometers. (d) At wave lengths below the blue maximum, the rate decreases progressively to 400 nanometers.     

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.     

Action spectra for polarotropism and phototropism in protonemata of the fern Adiantum capillus-veneris

The action spectrum for polarotropism was determined, using the Okazaki large spectrograph, by brief irradiation with light between 260 nm and 850 nm in single-celled protonemata of the fern Adiantum capillus-veneris L., which had been cultured for 6 days in red light and then in the dark for 15 h. The action spectrum had a peak at around 680 nm. This effect was nullified by subsequent irradiaton with far-red light, and typical red/far-red reversibility was observed, indicating the involvement of phytochrome. Polarized ultraviolet or blue light had no effect on the direction of apical growth. The action spectrum for phototropism was also determined in the red light region by means of brief microbeam irradiation of a flank of the subapical region of the protonema. This spectrum showed a peak at 662 nm which was consistent with the absorption peak of phytochrome, but not with the peak of the action spectrum for polarotropism.     

Enhancement of red-light-induced anthocyanin synthesis in sorghum first internodes by moderate low temperature given in the pre-irradiation culture period

Anthocyanin synthesis in the broom sorghum, Sorghum bicolor Moench cvs. Acme Broomcorn and Sekishokuzairai-Fukuyama, is mediated separately or synergistically by an ultraviolet light-B (UV-B) photoreceptor and phytochrome. When seedlings were exposed to moderate low temperatures ranging from 12 to 20° C before irradiation, only the phytochrome-mediated anthocyanin synthesis was markedly enhanced compared with the control, which was kept throughout at 24° C; synthesis mediated by the UV-B photoreceptor was unaffected. The effectiveness of an exposure to 20° C increased as the duration of exposure increased up to 24 h and as the time of exposure became closer to the time of irradiation. However, when seedlings were exposed to 20° C from after irradiation until harvest, anthocyanin syntheses induced by both UV-B and red light were equally suppressed, probably due to the general reduction of metabolism involved in anthocyanin synthesis that is a consequence of lower temperature. The results support the view that the signal transduction of the pyhtochrome system is different from that of the UV-B photoreceptor, and indicate that the phytochrome system may involve a step or steps which are amplified by a previous exposure to the moderate low temperature.Abbreviations FR far-red light - LT low temperature - MLT moderate low temperature - Pfr far-red-light-absorbing form of phytochrome - R red light - UV ultraviolet light - UV-B ultraviolet light-B We thank Drs. Y. Takeuchi (Shionogi Pharmaceutical Company, Aburahi, Shiga) and K. Hosaka (the Experimental Farm, Kobe University, Kasai) for seeds; Dr. M. Watanabe and Mr. M. Kubota (the National Institute for Basic Biology, Okazaki) for operation of the spectrograph. This work was supported by grants from the Yamada Science Foundation, Ministry of Education (No. 63480015 and 03454048), and the National Institute for Basic Biology (Large Spectrograph grant No. 91-523).     

Photocontrol of fungal spore germination

Germination of Puccinia graminis f. sp. tritici uredospores is inhibited by continuous irradiation. Prehydration of spores enhances both dark germination and photoinhibition. Simultaneous irradiation with ineffective red (653 nanometers) and inhibitory far red light (720 nanometers) results in partial nullification of the inhibition brought about by far red light alone. This result would be consistent with the involveent of a photoreversible pigment system similar to phytochrome, operating via the high irradiance reaction.     

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.     

Action Spectrum for Resetting the Circadian Phototaxis Rhythm in the CW15 Strain of Chlamydomonas: I. Cells in Darkness

We have developed protocols for phase shifting the circadian rhythm of Chlamydomonas reinhardtii by light pulses. This paper describes the photobiology of phase-resetting the Chlamydomonas clock by brief (3 seconds to 15 minutes) light pulses administered during a 24 hour dark period. Its action spectrum exhibited two prominent peaks, at 520 and 660 nanometers. The fluence at 520 nanometers required to elicit a 4 hour phase shift was 0.2 millimole photon per square meter, but the pigment that is participating in resetting the clock under these conditions is unknown. The fluence needed at 660 nanomoles to induce a 4 hour phase shift was 0.1 millimole photon per square meter, which is comparable with that needed to induce the typical low fluence rate response of phytochrome in higher plants. However, the phase shift by red light (660 nanometers) was not diminished by subsequent administration of far-red light (730 nanometers), even if the red light pulse was as short as 0.1 second. This constitutes the first report of a regulatory action by red light in Chlamydomonas.     

Control of Leaf and Stem Growth in Light-grown Pea Seedlings by Two High Irradiance Responses

The control exerted by light on leaf and stem growth in light-grown Alaska pea seedlings was studied during the main photoperiod. Two high irradiance responses were observed. The action spectrum for one had a single sharp peak at 600 nanometers. The action spectrum for the other showed a broad peak between 440 and 470 nanometers. These two light responses must be activated simultaneously for any inhibition of stem growth or promotion of leaf growth. Both action spectra may be explained in terms of the high irradiance response of phytochrome.     

Wavelength Effect on the Action of a N-Phenylimide S-23142 and a Diphenylether Acifluorfen-Ethyl in Cotyledons of Cucumber (Cucumis sativus L.) Seedlings

Specific wavelengths of light required for expression of phytotoxic activity of S-23142 (N-[4-chloro-2-fluoro-5-propargyloxy]phenyl-3,4,5,6-tetra- hydrophthalimide) and acifluorfen-ethyl (ethyl-5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitro benzoic acid) were determined in cotyledons of cucumber seedlings using the Okazaki Large Spectrograph. Leakage of amino acids from the cotyledons was measured as an indication of the phytotoxic activity. The wavelength effects showed common major peaks of activity at 550 and 650 nanometers and a minor peak at 450 nanometers for both herbicides, indicating a common primary photoreaction. Concomitant application of DCMU (3-[3,4-dichlorophenyl]-1,1-dimethylurea) with S-23142 had little influence on the effective wavelengths for S-23142 activity. Light of 450 and 650 nanometers was relatively less effective in achlorophyllous tissue grown in far red light than in green tissue. These results strongly suggest that the phytotoxic action of S-23142 and diphenylethers involves multiple photoreactions and that one of the photoreceptor pigments may be chlorophyll or its related pigment, although photosynthesis is not involved.     

Phytochrome-induced Increase of Fluorescein Translocation in Mung Bean Hypocotyls

Moderate doses of red (660 nanometer) irradiation cause a rapid increase in the translocation of fluorescein in dark-grown mung bean hypocotyl (Vigna radiata L.) segments. The increase fails to appear following large doses of red (660 nanometers) irradiation. The red induced increase is prevented by a subsequent far red (730 nanometer) irradiation. Reversibility suggests the participation of phytochrome in the process. The increase in translocation is attributed to the generation of a positive electrostatic charge in the plasma membrane by some action of phytochrome on membrane molecules.     

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°.     

Comparison of Photomorphogenic Responses to UV Light in Red and White Cabbage (Brassica oleracea L.)

Photoinhibition of hypocotyl growth in white cabbage (Brassica oleracea L., cv “Bianco Brunswick”) is controlled by UV absorbing receptor(s) and the phytochrome system, while in red cabbage (cv “Rosso Olandese tardivo invernale”) phytochrome can act without any requirement for the action of a specific UV receptor. Similar results have been obtained for the photoregulation of anthocyanin production. Twenty-four hour preirradiations with UV light or 692 nanometers light lead to the same increase in responsiveness of the system toward Pfr in a following dark period, suggesting a phytochrome promotion of subsequent light induction for both.