Modulation of a mitochondrial function by oat phytochrome in vitro

Previous data in the literature have indicated that phytochrome could alter the rate of reduction of exogenously added NADP by a pea mitochondrial preparation in vitro. These results could not be duplicated using a mitochondrial preparation isolated from etiolated oat seedlings. Further experimentation demonstrated that the addition of Pr to the preparation, in combination with a far red light illumination, could significantly reduce the rate of oxidation of NADH by the external dehydrogenases of oat mitochondria. This response was characterized by a 15% decrease in reaction velocity at saturating substrate concentrations and a 2-fold increase in apparent K_m as compared to values obtained after Pfr plus red light treatment. The response was photoreversible, the rate of oxidation of exogenous NADH being determined by the last light illumination given to the mitochondrial preparation. The interaction between phytochrome and the mitochondria was apparently occurring at the level of the inner mitochondrial membrane. A requirement for these results was that the mitochondria be isolated from plants that were illuminated with white or red light before extraction; mitochondria from unirradiated plants showed no dehydrogenase response to treatments with phytochrome plus actinic light.     

Short term phytochrome control of oat coleoptile and pea epicotyl growth

Continuous recordings of the effect of light on oat (Avena sativa L. cv. Victory) coleoptile and pea (Pisum sativum L. cv. Alaska) epicotyl growth were made. Using a single excised coleoptile 10 minutes of red light was found to promote growth after a latent period of 46 minutes. The stimulation was transient and was not far red-reversible. Blue and far red light also promoted growth with similar kinetics. The action of continuous red or far red light was similar to that of 10-minute light. The growth of the intact pea third internode (as well as excised segments) was strongly inhibited by red light, with a latent period of 80 minutes. This effect was far red-reversible, and far red and blue light caused only a slight inhibition of growth.     

Photoreversible calcium fluxes induced by phytochrome in oat coleoptile cells

The chromometallic dye murexide was used to measure photoreversible Ca fluxes in apical tips of etiolated oat coleoptiles and in suspension cultures of protoplasts derived from the coleoptile segments. Phytochrome presence in the protoplasts was indicated by a repeatably photoreversible ΔA(725 - 800 nm) of >0.001 A centimeters⁻¹, recorded on a dual wavelength spectrophotometer. Concentrations of Ca in the solution bathing the cells were observed to change photoreversibly, red irradiation inducing an increase in the medium Ca concentration and subsequent farred irradiation inducing a decrease down to near dark control levels. These changes could be measured in media with or without exogenously added Ca. Protoplasts from green primary leaves of oat, which had no spectro-photometrically detectable phytochrome, showed no photoreversible Ca fluxes when measured by the same method. These data imply that red light induces an efflux of Ca from phytochrome-containing cells and that far red light can reverse this change by promoting a Ca reentry into these cells.     

Phytochromabhängiger Ionentransport in Erbsensämlingen

Summary Five to 6 day old dark-adapted dwarf and tall pea seedlings grown in water culture were illuminated for ten minutes with red light and/or ten minutes with far-red light, and 90 to 170 minutes later their roots were immersed in a 0.2 mM K⁺ solution containing labeled ⁸⁶Rb⁺. After two hours uptake the fresh-weights and radioactivities of the shoot organs were determined. It was found that red light inhibits K⁺uptake into internodes and promotes uptake into the plumula. The red-light effect on K⁺transport precedes the red-light induced growth inhibition of internodes and growth promotion of leaves and is abolished by far-red light given immediately after red. The red-light effect on K⁺transport is independent of the concentration of K⁺ given to the roots in the range between 0.2 to 125 mM.     

Apparent Pfr killer reaction and P* denaturation in segments of etiolated pea epicotyl

When totally etiolated pea epicotyls were cut into segments and incubated with potassium phosphate buffer, pH 6.0, in the dark at 25 C, an instantaneous loss of photoreversible absorbance change, Δ (ΔA) between 660 and 730 nm, was observed after the first irradiation with actinic red light in the spectrophotometric measurement of phytochromein vivo. The shorter the epicotyl segments, and the longer the period of dark incubation, the greater was the loss detected in the measurement. A remarkable decline of Δ(ΔA) in the far-red region was seen inin vivo difference spectra for phytochrome, after the epicotyl segments were incubated in the dark at 25 C. As the period of dark incubation was prolonged, the ratio of the maximal change of Δ(ΔA) in the far-red region to that in the red region was reduced. It decreased to ca. one third of the initial value after incubation for 8 hr. The evidence indicates that Pfr killer activity and P^* denaturation, both of which have so far been known onlyin vitro, can also occur in segments of etiolated pea epicotyls.     

Phytochrome control of two low-irradiance responses in etiolated oat seedlings

Light-induced coleoptile stimulation and mesocotyl suppression in etiolated Avena sativa (cv. Lodi) has been quantitated. Etiolated seedlings showed the greatest response to light when they were illuminated 48 to 56 hours after imbibition. Two low-irradiance photoresponses for each tissue have been described. Red light was 10 times more effective than green and 1,000 times more effective than far red light in evoking these responses. The first response, which resulted in a 45% mesocotyl suppression and 30% coleoptile stimulation, had a threshold at 10⁻¹⁴ einsteins per square centimeter and was saturated at 3.0 × 10⁻¹² einsteins per square centimeter of red light. This very low-irradiance response could be induced by red, green, or far red light and was not photoreversible. Reciprocity failed if the duration of the red illumination exceeded 10 minutes. The low-irradiance response which resulted in 80% mesocotyl suppression and 60% coleoptile stimulation, had a threshold at 10⁻¹⁰ einsteins per square centimeter and was saturated at 3.0 × 10⁻⁸ einsteins per square centimeter of red light. A complete low-irradiance response could be induced by either red or green light but not by far red light. This response could be reversed by a far red dose 30 times greater than that of the initial red dose for both coleoptiles and mesocotyls. Reciprocity failed if the duration of the red illumination exceeded 170 minutes. Both of these responses can be explained by the action of phytochrome.     

Photosensibilité des graines depinus banksiana Lamb.

Germination ofPinus banksiana seeds is controlled by the photoreversible phytochrome reaction. The seeds, even unimbibed, are sensitive to red light. At 660 nm, the energy required to promote germination to the same order of magnitude is much higher for unimbibed seeds than for the imbibed ones. In both cases it is possible to reverse the effect of a single red light irradiation by applying far red light (730 nm).     

Phytochrome Controlled Adhesion of Mung Bean Root Tips to Glass: A Detailed characterization of the Phenomenon

Various parameters of the Tanada effect (Proc. Natl. Acad. Sci. U.S. 59: 376–380. 1968) have been defined. This phenomenon, in which root tips of Phaseolus aureus L. adhere to a negatively charged glass surface when they are irradiated with 660 nm (red) light and release under 730 nm (far-red) light, has been characterized as follows. Secondary roots, whether etiolated or light grown exhibit photoreversible adhesion. Primary roots do not. Tips from 6–8 mm secondary roots exhibit the best response to red light, whereas tips from 3 mm roots respond best to far-red light. Red light saturetes the adhesion system at about 50 μ W/cm²xnm and far-red light, release system at about 150 ü W/cm² xnm. The adhesion effect begins to show escape from far-red reversibility within 60–90 seconds, an observation quite different from other “typical” long term de- etiolation effects. In addition, root tips irradiated with red light begin to release spontaneously in the dark after 10 min, and have nearly completed release after 50 min. Tips irradiated with continuous red light show gradual release after 15 minutes of exposure. Whether these data indicate an extremely rapid dark reversion of P_fr to P_r or decay of P_fr under continuous red light is not known at this time. In order to study tip adhesion and release, the glass beaker surface may be negatively charged with thiocyanate (SCN^-), nitrate (NO₃^-), sulfate (SO₄^2-), chloride (Cl^-), phosphate (PO₄^3-), citrate (C₆H₅O₇^3-), oxalate (C₂O₄^2-) or glutamine (C₅H₈NO₄^-). Benzoate (C₇H₅O₂^-) and acetate (CH₃COO^-) were found to be relatively ineffective for red light adhesion, however when citrate and oxalate were used release was inhibited. This was apparently due to a chelation of Ca²⁺since release began immediately as excess Ca⁺² was added to the bathing solution. Substitution of GTP, ITP, UTP, or CTP for ATP resulted in only 20 to 40% adhesion and release for GTP, ITP and UTP, CTP showed normal adhesion kinetics under red light but very slow release kinetics under far-red light. The effects of red and far-red light in the numbers of secondary roots are that red light inhibits root initiation while far-red light partially reverses the red light effect.     

Action spectra for C-glucosylflavone accumulation in Hordeum vulgare plumules

Five-day-old etiolated barley plumules contain the C-glucosylflavones saponarin, lutonarin, and lutonarin 3′-methyl ether. When harvested 24 hr after illumination, increased flavonoid levels were essentially linear with increased energies of monochromatic light at seven wavelengths between 450 and 750 nm. Action spectra for saponarin and for a mixture of lutonarin and its 3′-methyl ether were determined between 380 and 760 nm at 6.6 kerg·cm^?2. The saponarin action spectrum showed distinct peaks at 620 and at 660 nm. These two peaks were similar in their photoreversibility when followed by either 6·6 or 34 kerg·cm^?2 of far-red light. Phytochrome is apparently the photoreceptor for the saponarin action spectrum. Lutonarin and its 3′-methyl ether showed peaks at 520 580, 620 and near 660 nm. The 660 nm peak was not photoreversible by 6·6 kerg·cm^?1, but was by 34 kerg·cm_?2, of far-red light. Phytochrome and protochlorophyll are the likely photoreceptors for these 3′-substituted flavonoids.     

Photoreversible and photoirreversible absorbance changes in the red and far-red spectral regions in Zea primary roots

When 2-mm apical segments of the primary roots of Zea mays L.(cv. Golden Cross Bantam 70) were irradiated successively withred and far-red light, a photoirreversible absorbance decreasewas separated from the red far-red reversible absorbance changetypical of phytochrome. The difference spectrum of the reversiblechange showed maximum absorbance changes at 666 and 730 nm,while the photoirreversible change induced by red light showeda maximum decrease at 640 nm. The photoreversible absorbancechange was linearly proportional to the fluence of red lightbetween 1 and 6 J m^–2, while the photoirreversible absorbancechange was proportional to its logarithm. Red light of approximately6 J m^–2 induced 50% of the maximum photoirreversible absorbancechange at 640 nm but only about 25% of the maximum photoreversibleabsorbance change. Moreover, no effect of ascorbate on the twoabsorbance changes was observed. ¹Faculty of Education, University of Yamagata, Yamagata 990,Japan. (Received November 2, 1980; )     

Photoreversible and photoirreversible absorbance changes in the red and far-red spectral regions in Zea primary roots

When 2-mm apical segments of the primary roots of Zea mays L.(cv. Golden Cross Bantam 70) were irradiated successively withred and far-red light, a photoirreversible absorbance decreasewas separated from the red far-red reversible absorbance changetypical of phytochrome. The difference spectrum of the reversiblechange showed maximum absorbance changes at 666 and 730 nm,while the photoirreversible change induced by red light showeda maximum decrease at 640 nm. The photoreversible absorbancechange was linearly proportional to the fluence of red lightbetween 1 and 6 J m^–2, while the photoirreversible absorbancechange was proportional to its logarithm. Red light of approximately6 J m^–2 induced 50% of the maximum photoirreversible absorbancechange at 640 nm but only about 25% of the maximum photoreversibleabsorbance change. Moreover, no effect of ascorbate on the twoabsorbance changes was observed. ¹Faculty of Education, University of Yamagata, Yamagata 990,Japan. (Received November 2, 1980; )     

Equivalence between Pfr and Cyclic AMP in the Induction of d-Usnic Acid Dehydrogenase in the Lichen Evernia prunastri

d-Usnic acid dehydrogenase is induced in Evernia prunastri thalli by a supply of exogenous d-usnic acid in light. This effect is enhanced by red light pulses through a two step way: a very rapid increase of activity after the first 10 minutes of red light, which is not reversed by far-red light, and a slow enhancement following successive red light pulses at the beginning of each hour of incubation. The last response is completely reversed by far-red following red light. Although induction of the enzyme is not achieved in the dark, 0.1 and 0.5 millimolar cyclic AMP, or 0.1 millimolar dibutyryl cyclic AMP substitutes light action and, then, the enzyme is produced. In addition, phytochrome—far red-absorbing form—increases the amount of endogenously produced cyclic AMP and this effect is shown to be photoreversible when ethylenediaminetetraacetic acid is inhibiting adenylate cyclase.     

Regulation of swelling of etiolated-wheat-leaf protoplasts by phytochrome and gibberellic acid

The effect of light on the size of intact protoplasts isolated from the primary leaves of etiolated Triticum aestivum was studied. A 2-min red-light irradiation in the presence of 1 mM KCl was sufficient to cause a swelling of protoplasts compared with those maintained in darkness. The effect was photoreversible by far-red light over two light cycles, indicating the involvement of phytochrome. At 4°C, escape from reversibility occurred between 2 and 5 min after the exposure to red light. In exposure-response experiments, 20 s red light at 27 μmol m^-2s^-1 was sufficient to saturate the response. Exogenous gibberellic acid added in darkness in the presence of KCl also induced protoplast swelling. Gibberellins may act as an intermediate in the phytochrome-induced swelling of protoplasts.     

Gibberellic Acid-Promoted Lignification and Phenylalanine Ammonia-lyase Activity in a Dwarf Pea (Pisum sativum)

The effects of gibberellic acid on lignification in seedlings of a dwarf and a tall cultivar of pea (Pisum sativum) grown under red or white light or in the darkness, were studied. Gibberellic acid (10⁻⁶-10⁻⁴ m) promoted stem elongation in both light and dark and increased the percentage of lignin in the stems of the light-grown dwarf pea. The gibberellin had no effect on the lignin content of the tall pea although high concentrations (10⁻⁴ m) promoted growth of the tall plants. Time course studies indicated that the enhanced lignification in the gibberellin-treated dwarf plants occurred only after a lag period of several days. It was concluded that gibberellic acid-enhanced ligmification had no direct relation to gibberellic acid-promoted growth. The activity of phenylalanine ammonia-lyase (E.C. 4.3.1.5) was higher in gibberellin-treated dwarf plants grown under white or red light than in untreated dwarf plants. Gibberellic acid had no detectable effect on the activity of this enzyme when the plants were grown in darkness, just as it had no effect on lignification under dark conditions. The data suggest that in gibberellin-deficient peas the activity of phenylalanine ammonia-lyase is one of the limiting factors in lignification.     

Reversible phytochrome regulation influenced the severity of ozone-induced visible foliar injuries in Trifolium subterraneum L.

In Trifolium subterraneum, oxidative stress caused by ozone has been shown to result in more severe visible foliar injuries when plants were kept in dim broadband white light during the night (i.e. a long photoperiod) compared to darkness during the night (a short photoperiod). As phytochrome signalling is involved in photoperiod sensing, the effect of night-time red and far-red illumination on the ozone-induced response was studied. T. subterraneum plants were treated with ozone enriched air (70?ppb) for either 1?h for a single day or 6?h for three consecutive days. After the first ozone exposure, plants were separated into six night-time light regimes during the two subsequent nights (10?h?day, 14?h night): (1) darkness, (2) far-red light (FR), (3) a short night-break of red followed by far-red light during an otherwise dark night (R FR), (4) a short night-break of red, far-red and finally red light during an otherwise dark night (R FR R), (5) dim white light (L) and (6) red light (R). The treatments L and R resulted in significantly more severe ozone-induced visible foliar injuries relative to D and FR treatments, indicating a phytochrome-mediated response. The night-breaks resulted in a photoreversible and significantly different ozone response depending on the light quality of the last light interval (R FR or R FR R), supporting a photoreversible (between P_r and P_fr) phytochrome signalling response. Thus, in T. subterraneum, the outcome of oxidative stress due to ozone appears to depend on the photoperiod mediated by the night-time conformation of phytochrome.     

Oxaloacetate translocator in plant mitochondria

(1) Mitochondria were prepared from leaves of spinach, green and etiolated seedlings and roots of pea, potato tuber and rat liver and heart. In the case of leaf mitochondria, an improved isolation procedure resulted in high respiratory rates (460–510 nmol/mg protein per min) and good respiratory control ratio (6.8–9.8) with glycine as substrate. (2) In these mitochondria oxaloacetate transport was studied either by following the inhibitory effect of oxaloacetate on the respiration of NADH-linked substrates or by determining the consumption of [4-¹⁴C]oxaloacetate. (3) Studies of the competition by other carboxylates and effect of inhibitors on the oxaloacetate transport demonstrate that mitochondria from spinach leaves, green pea seedlings, etiolated pea seedlings and pea roots contain a specific translocator for oxaloacetate with a very high affinity to its substrate (K_m = 3–7 μM) and an even higher sensitivity to its competitive inhibitor phthalonate (K_i = 3–5 μM). The V_max values ranged from 150 to 180 nmol/mg protein per min for mitochondria from etiolated pea seedlings and pea roots and from 550 to 570 nmol/mg protein per min for mitochondria from spinach leaves and green pea seedlings. In mitochondria from potato tuber, the K_m was about one order of magnitude higher (V_max = 450 nmol/mg protein per min). In mitochondria from rat liver and rat heart, a specific translocator for oxaloacetate was not found. (4) The oxaloacetate translocator enables the functioning of a malate-oxaloacetate shuttle for the transfer of reducing equivalents across the inner mitochondrial membrane. (5) This malate-oxaloacetate shuttle appears to play a role in the photorespiratory cycle in catalyzing the transfer of reducing equivalents generated in the mitochondria during glycine oxydation to the peroxysomal compartment for the reduction of β-hydroxypyruvate. (6) Interaction between the mitochondrial and the chloroplastic malate oxaloacetate shuttles would make it possible for surplus-reducing equivalents, generated by photosynthetic electron transport, to be oxidized by mitochondrial electron transport.     

Red light and auxin effects on rubidium uptake by oat coleoptile and pea epicotyl segments

Apical segments of etiolated oat (Avena sativa L. cv. Victory) coleoptiles showed enhanced uptake of [⁸⁶Rb⁺] when tested 30 minutes after a 5-minute red irradiation. The response was partly reversible by far red light. Uptake was sensitive to carbonyl cyanide m-chlorophenyl hydrazone, but not to isotonic mannitol. Indoleacetic acid (10⁻⁷ molar) caused a very pronounced and rapid stimulation of uptake. Basal coleoptile segments also exhibited a red light-enhanced uptake, but not an effect of red light on changes in the pH of the medium. The [⁸⁶Rb⁺] uptake of third internode segments from etiolated peas (Pisum sativum L. cv. Alaska) was not affected by either red light or auxin. This tissue also showed no red light effect on acidification of the medium. It is concluded that alteration of [⁸⁶Rb⁺] flux is not a general feature of phytochrome action.     

The origin and turnover of organelle membranes in castor bean endosperm

The origin and turnover of organelle membranes in castor bean (Ricinus communis L. var. Hale) endosperm was examined using choline-¹⁴C as a phospholipid precursor. On sucrose gradients three major particulate fractions were separated; a light membranous fraction (density 1.11-1.13 gram per cm³), the mitochondria (1.18 gram per cm³), and the glyoxysomes (1.24 gram per cm³). Choline-¹⁴C was readily incorporated into lecithin in all three particulate fractions, but the light membranous fraction became labeled first. Incorporation continued into all three fractions for 6 hours, at which time the available choline-¹⁴C had been completely used. Subsequently, ¹⁴C was lost from the three components at distinctly different rates. When an excess of unlabeled choline was added after 1 hour (pulse-chase experiment), incorporation of choline-¹⁴C into glyoxysomes and mitochondria continued for three hours, but at a diminishing rate. This was followed by a period in which the ¹⁴C content of the mitochondria declined at a rate expected, if the half life of lecithin in the membrane were about 50 hours and that of the glyoxysomes 10 hours. These values are close to those calculated from the experiments in which no chase was used. The labeling in the light membrane fraction behaved differently from that of the mitochondria and glyoxysomes following the chase of unlabeled choline. Incorporation continued for only 1 additional hour, and then the ¹⁴C content declined sharply in the subsequent 4 hours. The early kinetics and subsequent interrelationships are those expected if the lecithin in the membranes of mitochondria and glyoxysomes originates in components of the light membrane fraction.     

Genetic Regulation of Development in Sorghum bicolor: VI. The ma(3) Allele Results in Abnormal Phytochrome Physiology

Physiological processes controlled by phytochrome were examined in three near-isogenic genotypes of Sorghum bicolor, differing at the allele of the third maturity gene locus. Seedlings of 58M (ma₃^R ma₃^R) did not show phytochrome control of anthocyanin synthesis. In contrast, seedlings of 90M (ma₃ma₃) and 100M (Ma₃Ma₃) demonstrated reduced anthocyanin synthesis after treatment with far red and reversal of the far red effect by red. De-etiolation of 48-hour-old 90M and 100M dark-grown seedlings occurred with 48 hours of continuous red. Dark-grown 58M seedlings did not de-etiolate with continuous red treatment. Treatment of seedlings with gibberellic acid or tetcyclacis, a gibberellin synthesis inhibitor, did not alter anthocyanin synthesis. Levels of chlorophyll and anthocyanin were lower in light-grown 58M seedlings than in 90M and 100M. Etiolated seedlings of all three genotypes have similar amounts of photoreversible phytochrome. Crude protein extracts from etiolated seedlings were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose. Phytochrome was visualized with Pea-25, a monoclonal antibody directed to phytochrome from etiolated peas. The samples from all three genotypes contained approximately equivalent amounts of a prominent, immunostaining band at 126 kD. However, the sample from 58M did not show a fainter, secondary band at 123 kD that was present in 90M and 100M. The identity and importance of this secondary band at 123 kD is unknown. We propose that 58M is a phytochrome-related mutant that contains normal amounts of photoreversible phytochrome and normal phytochrome protein when grown in the dark.