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.     

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.     

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.     

Ultraviolet action spectrum for anthocyanin formation in broom sorghum first internodes

An action spectrum for anthocyanin formation in dark-grown broom sorghum (Sorghum bicolor Moench, cv Acme Broomcorn and cv Sekishokuzairai Fukuyama Broomcorn) seedlings was determined over the wavelength range from 260 to 735 nanometers. The action peaks were at 290, 650, 385, and 480 nanometers in descending order of height. The action of the 290-nanometer peak was not affected by subsequently given far red light, whereas those of the other three action peaks were nullified completely. The nullification of the 385-nanometer peak action by far red light was reversible. When an irradiation at these action peaks was followed by a phytochrome-saturating fluence of red light irradiation, the action of the 290-nanometer peak remained, whereas that of the 385-nanometer peak as well as those of the 650- and 480-nanometer peaks was masked by the action of the second irradiation. These findings suggested that the 290- and 385-nanometer action peaks involved different photoreceptors, the latter being phytochrome. The blue light-absorbing photoreceptor as reported to be a prerequisite for phytochrome action in milo sorghum was not found to exist in the broom sorghums.

The action spectrum deprived of the involvement of phytochrome was determined in the ultraviolet region by irradiating with far red light following monochromatic ultraviolet light. The spectrum had a single intense peak at 290 nanometers and no action at all at wavelengths longer than 350 nanometers.

     

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.     

Inhibition of phytochrome synthesis by gabaculine

Gabaculine (5-amino-1,3-cyclohexadienylcarboxylic acid), a transaminase inhibitor, also inhibits chlorophyll formation in plants, and the effect of this compound can be counteracted by 5-aminolevulinic acid (ALA) (Flint, personal communication, 1984). Since it is probable that ALA also serves as a precursor to phytochrome, the effects of gabaculine on phytochrome synthesis in developing etiolated seedlings were examined using in vivo spectrophotometry. Preemergence treatment with gabaculine was found to inhibit initial phytochrome synthesis in peas (Pisum sativum L.), corn (Zea mays L.), and oats (Avena sativa L.). In general, reduction in phytochrome correlated with reduction in chlorophyll. However, the extent of inhibition of phytochrome synthesis was not as great as that of chlorophyll synthesis, perhaps due to preexisting phytochrome in the seed. Foliar treatment of etiolated pea seedlings prior to light-induced destruction of phytochrome inhibited subsequent phytochrome resynthesis in the dark. These results suggest that both initial synthesis and resynthesis of phytochrome require de novo synthesis of chromophore as well as apoprotein.     

Far-red light-induced changes in intracellular potentials of spinach mesophyll cells: interaction with red light

In green plants, the large bioelectric changes that photosynthetically active light stimulates make it difficult to observe electrical potential changes related to phytochrome photoconversion. As a first step towards distinguishing between photosynthetic and phytochrome effects, we showed that red light enhances far-red stimulated intracellular potential changes in spinach (Spinacia oleracea) leaf mesophyll cells.

For a dark-adapted leaf, the response to far-red light increased during the first 10 to 30 exposures of 2.5 minutes, after which it was constant. The intracellular potential depolarized by an average of 0.3 millivolts during each 2.5-minute far-red light period, and returned to the resting value during each subsequent dark period. Continuous supplementary red light (at 1-5% of the fluence rate of the far-red light that stimulated the depolarizations) increased the response to far-red 2- to 3-fold. Supplementary red light did not amplify the response to alternating 702 nanometers light and dark periods. The Emerson enhancement effect thus does not seem to explain amplification of the response to 730 nanometers light by supplementary red light. This does not prove that photosynthetic pigments are not involved in some other way.

     

Characterization of Tobacco Expressing Functional Oat Phytochrome : Domains Responsible for the Rapid Degradation of Pfr Are Conserved between Monocots and Dicots

Constitutive expression of a chimeric oat phytochrome gene in tobacco (Nicotiana tabacum) results in the accumulation of a functional 124-kilodalton photoreceptor that markedly alters the phenotype of light-grown tobacco (Keller et al. [1989] EMBO J 8: 1005-1012). Here, we provide a detailed phenotypic and biochemical characterization of homozygous tobacco expressing high levels of oat phytochrome. Phenotypic changes include a substantial inhibition of stem elongation, decreased apical dominance, increased leaf chlorophyll content, and delayed leaf senescence. Oat phytochrome synthesized in tobacco is indistinguishable from that present in etiolated oats, having photoreversible difference spectrum maxima at 665 and 730 nanometers, exhibiting negligible dark reversion of phytochrome—far red-absorbing form (Pfr) to phytochrome—red-absorbing form (Pr), and existing as a dimer with an apparent size of approximately 300 kilodaltons. Heterodimers between the oat and tobacco chromoproteins were detected. Endogenous tobacco phytochrome and transgenically expressed oat phytochrome are rapidly degraded in vivo upon photoconversion of Pr to Pfr. Breakdown of both oat and tobacco Pfr is associated with the accumulation of ubiquitin-phytochrome conjugates, suggesting that degradation occurs via the ubiquitin-dependent proteolytic pathway. This result indicates that the factors responsible for selective recognition of Pfr by the ubiquitin pathway are conserved between monocot and dicot phytochromes. More broadly, it demonstrates that the domain(s) within a plant protein responsible for its selective breakdown can be recognized by the degradation machinery of heterologous species.     

Some spectral properties of pea phytochrome in vivo and in vitro

The transformation difference spectrum for phytochrome (Pr spectrum minus Pfr spectrum) in pea tissue is determined below 560 nanometers and compared with similar data on phytochrome in vitro The difference spectrum in vivo between phytochrome intermediates and Pfr is also shown for comparison with the data on phytochrome solutions. These comparisons show that the peaks in the spectra occurring in the blue wave lengths are shifted to shorter wave lengths and are much enhanced when phytochrome is extracted from the cell and placed in solution. The results indicate that the physicochemical state of phytochrome in the cell may be different from that of the extracted pigment.     

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.     

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.     

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.     

Phytochrome activation of k channels and chloroplast rotation in mougeotia: action spectra

The action spectra for K⁺ channel activation and chloroplast rotation are shown to be similar. Both phenomena exhibit activation at 660 nanometers, inhibition at 740 nanometers, and partial activation at 460 to 500 nanometers. This confirms that K⁺ channels in Mougeotia are regulated by phytochrome, and indicates that both phenomena share at least part of the same transduction pathway.     

Photomorphogenesis in Arabidopsis thaliana (L.) Heynh: Threshold Intensities and Blue-Far-red Synergism in Floral Induction

Arabidopsis seeds were germinated on sterile mineral agar supplemented with 1% glucose and cultured under continuous light regimes. With 4-hour incandescent plus 20-hour monochromatic illumination in the region from 400 to 485 nanometers there was effective floral induction at an intensity of 100 microwatts per square centimeter. Exclusion of far red wave lengths from the 4-hour incandescent period sharply reduced the effectiveness of subsequent monochromatic blue light in promoting floral induction. Delayed floral induction occurred under continuous incandescent light lacking far red and was attributable to the blue wave lengths. Continuous 485 nanometer (100 microwatts per square centimeter) exposure without any white light treatment during the postgermination growth period was ineffective in floral induction and meristem development. Light at 730 nanometers under the same conditions was partially effective, whereas energy between 500 and 700 nanometers was completely ineffective. When continuous monochromatic light at a 3-fold higher energy level was administered, all photomorphogenic responses were accomplished with 485 nanometer light, including germination and 100% floral induction without any white light treatment at any time during the experiment. Almost equal quantum effectiveness was calculated when equivalent quantum flux densities in the region from 710 to 740 nanometers or at 485 nanometers were used. It is postulated that floral induction in Arabidopsis may be the result of a continuous excitation of a stable form of far red-absorbing phytochrome localized in or on a membrane, and that excitation can be either by direct absorption of energy by far red-absorbing phytochrome or by transfer from an accessory pigment.     

Effect of far-red and green irradiation on the nyctinastic closure of albizzia leaflets

The nyctinastic closing of Albizzia julibrissin pinnules is delayed by exposure to far-red radiation at 710 and 730 nanometers, with the former more effective than the latter. Far-red radiation at 750 and 770 nanometers has no effect on the process. Red light at 660 nanometers, which by itself has no effect, delayed closure when given before or simultaneously with far-red radiation at 750 or 770 nanometers. Low doses of green light, on the other hand, prevented all far-red radiations from delaying closure when given together with one of them. Effectiveness peaks at 550 nanometers. Green light by itself has no effect on the closing process.

From these and previous results, it is concluded that phytochrome is one of two photoreceptors in the process, that the other photoreceptor is an unknown pigment, and that the unknown photoreceptor requires some prior effect of the far-red-absorbing form of phytochrome before its action. Predictions are made of some of the properties of the unidentified pigment.

     

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.     

Action spectrum for light-induced formation of adventitious shoots in hairy roots of horseradish

An action spectrum was determined for lightinduced formation of adventitious shoots in hairy roots of horseradish (Armoracia rusticana Gaert., Mey. et Scherb.) cultured in vitro. Near ultraviolet (350–400 nm), blue (440–460 nm) and red light (600–680 nm) were most effective in inducing formation of adventitious buds. Farred light (730 nm) inhibited the promotive effect of all three wavelength regions. These results are consistent with induction by phytochrome(s) of adventitious shoots in hairy roots of horseradish.     

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.     

An Action Spectrum for Light-induced Primordium Formation in a Basidiomycete, Favolus arcularius (Fr) Ames

The action spectrum for the initiation of fruiting (primordium formation) in Favolus arcularius was determined on the equal response basis. The detectable effect of light was observed in the region between 350 to 560 nanometers, showing six distinct peaks at 374, 398, 424, 446, 480, and 514 nanometers. The half maximum response is reached with 1.8 × 10⁸ ergs per cm² at the most effective wavelength, 398 nanometers. Since the inhibitors, diphenylamine and quinacrine, had no consistent effect on the primordium formation, it is suggested that the possible photoreceptor pigment(s) may be neither carotenoid nor flavinoid.     

Spectral Characterization and Proteolytic Mapping of Native 120-Kilodalton Phytochrome from Cucurbita pepo L

A spectral, immunochemical, and proteolytic characterization of native 120-kilodalton (kD) phytochrome from Cucurbita pepo L. is presented and compared with that previously reported for native 124-kD phytochrome from Avena sativa. The molecule was partially purified (~200-fold) in the phytochrome—far red-absorbing form (Pfr) in the presence of the protease inhibitor, phenylmethylsulfonyl fluoride, using a modification of the procedure initially developed to purify 124-kD Avena phytochrome. The spectral properties of the preparations obtained are indistinguishable from those described for 124-kD Avena phytochrome, including a Pfr λ_max at 730 nanometers, a spectral change ratio (ΔA_r/ΔA_fr) of 1.05, and negligible dark reversion of Pfr to the red-absorbing form (Pr) in the presence or absence of sodium dithionite. This lack of dark reversion in vitro contrasts with observations that Cucurbita phytochrome, like phytochrome from most other dicotyledons, exhibits substantial dark reversion in vivo. Ouchterlony double immunodiffusion analysis with polyclonal antibodies indicates that 120-kD Cucurbita phytochrome is immunologically dissimilar to 124-kD Avena phytochrome. However, despite this dissimilarity, immunoblot analyses of proteolytic digests have identified at least three spatially separate epitopes that are common to both phytochromes. Using endogeneous protease(s), a peptide map for Cucurbita phytochrome has been constructed and the role that specific domains play in the overall structure of the photoreceptor has been examined. One domain near the NH₂ terminus is critical to the spectral integrity of the molecule indicating that this domain plays a structural role analogous to that of a domain near the NH₂ terminus of Avena phytochrome. Proteolytic removal of this domain occurs preferentially in Pr and its removal shifts the Pfr λ_max to 722 nm, increases the spectral change ratio to 1.3, and substantially enhances the dark reversion rate. The apparent conservation of this domain among evolutionarily divergent plant species and its involvement in a conformational change upon photoconversion makes it potentially relevant to the mechanism(s) of phytochrome action. Preliminary evidence from gel filtration studies suggests that the 55-kD chromophoreless COOH-terminal region of the polypeptide contains a domain responsible for dimerization of phytochrome monomers.