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.     

Phytochrome-mediated Carotenoids Biosynthesis in Ripening Tomatoes

Red light induced and far red light inhibited carotenoid biosynthesis in ripening tomatoes (Lycopersicon esculentum Mill.) when compared to controls kept in the dark. Red illumination following far red illumination reversed the inhibitory action of far red light on carotenoid biosynthesis, suggesting a phytochrome-mediated process. Quantitation of individual carotenoids favored the hypothesis of two separate carotenoid biosynthetic pathways in tomatoes.     

The Effect of Red Irradiation on Plastid Ribosomal RNA Synthesis in Dark-grown Pea Seedlings

Dark-grown pea seedlings (Pisum sativum L.) were irradiated for a short period each day with low intensity red light (662 nm), red light immediately followed by far red light (730 nm), or far red light alone. Other plants were transferred to a white light regime (14 hours light/10 hours dark). There was no change in the amount of RNA in the tissue on a fresh weight basis after the various treatments. However, compared with dark-grown seedlings, those plants irradiated with red light showed an increase in the net RNA content per stem apex. In addition there was a two- to three-fold increase in ribosomal RNA of the etioplasts relative to the total ribosomal RNA. These increases were comparable to those found in plants grown in the white light regime. The changes were much smaller if the dark-grown plants were irradiated either with red light followed by far red light, or with far red light alone. Thus continuous light is not essential for the production of ribosomal RNA in plastids, and the levels of ribosomal RNA found in chloroplasts can also be attained in etioplasts of pea leaves in the dark provided the leaf phytochrome is maintained in its active form.     

Formation of chlorophyll B, and the fluorescence properties and photochemical activities of isolated plastids from greening pea seedlings

Chlorophyll b was first detectable after 10 minutes of illumination of etiolated pea seedlings (Pisum sativum L. var Greenfeast) with continuous white light. The chlorophyll a/b ratio decreased from 300 at 10 minutes to 15 after 1 hour. There was little change in the chlorophyll a/b ratio between 1 and 2 hours, and it declined to 3 between 2 and 5 hours of illumination. In red light, the time courses of total chlorophyll synthesis and chlorophyll a/b ratio were similar to those in white light for the first 5 hours of illumination. But with increasing time of illumination with red light, there was an increase in the chlorophyll a/b ratio to 7 after 30 hours. Illumination with white light of very low intensity also gave high chlorophyll a/b ratios. Seedlings which had been illuminated for varying periods and then returned to darkness always showed an increase in chlorophyll a/b ratio during the dark period. It is concluded that the synthesis of chlorophyll b is controlled by light.     

Photomorphogenesis in Sinningia speciosa, cv. Queen Victoria II. Stem Elongation: Interaction of a Phytochrome Controlled Process and a Red-requiring, Energy Dependent Reaction

When Sinningia plants were grown with fluorescent light of photosynthetic intensity for 8 hours each day, stems became abnormally elongated when the P_FR level was lowered by far red light given during the last half of several consecutive nights. However, plants were even taller if the source also emitted red light. Elongation was independent of the red/far red energy ratio if it was lower than one, but dependent upon irradiance at all values tested.

Elongation of plants irradiated by a well filtered far red source was presumed to be limited by a shortage of respiratory substrate. Enhancement by radiation shorter than 700 mμ was attributed to promotion of processes leading to increased substrate supply. Protochlorophyllide was regarded as the primary photoreceptor. Its photoreduction promoted chlorophyll synthesis which, in turn, increased photosynthetic capacity and thus substrate supply.

     

Phytochrome action on the timing of cell division in adiantum gametophytes

When filamentous protonemata of Adiantum capillus-veneris L. precultured under continuous red light were transferred to the dark, the apical cell divided about 24 to 36 hours thereafter. The time of the cell division was delayed for several hours by a brief exposure to far red light given before the dark incubation. The effect of far red light was reversed by a small dose of red light given immediately after the preceding far red light. The effects of red and far red light were repeatedly reversible, indicating that the timing of cell division was regulated by a phytochrome system. When a brief irradiation with blue light was given before the dark incubation, the cell division occurred after 17 to 26 hours in darkness. A similar red far red reversible effect was also observed in the timing of the blue light-induced cell division. Thus, the timing of cell division appeared to be controlled by phytochrome and a blue light-absorbing pigment.     

Photoregulation of Nicotinamide Adenine Dinucleotide Kinase Activity in Cell-free Extracts

This article gives evidence that NAD kinase activity is controlled by the action of phytochrome. The NADP level rapidly increased in the cotyledons of seedlings of Pharbitis nil strain Violet (a short day plant), when the inductive dark for flowering was interrupted with a 5-minute illumination of red light. Illumination with far red light immediately after illumination with red light counteracted partly the effect of the latter.     

Phytochrome Action in Oryza sativa L: IV. Red and Far Red Reversible Effect on the Production of Ethylene in Excised Coleoptiles

Excised apical segments of etiolated rice (Oryza sativa L.) coleoptiles produced ethylene. Increasing the number of cut sites per coleoptile increased the rate of ethylene formation. Ethylene produced by an etiolated-intact seedling in the dark was about a half of that by the excised coleoptile segment. Red light of low energy as well as of continuous irradiation inhibited the production of ethylene. The inhibition by a low energy dose of red light was partly relieved, if the red light was followed immediately by a small dose of far red light. The effect of red and far red light was repeatedly reversible, indicating that ethylene production was regulated by a phytochrome system. If the exposure to far red light was preceded by a period of darkness, this photoreversibility disappeared; 50% of the initial reversibility was lost within 5 hours. Applied ethylene (10 microliters per liter) significantly promoted the growth of intact coleoptiles of either totally etiolated or red light-treated seedlings, but had no effect on the excised apical segment of coleoptile.     

Annual Variation in the Effect of Red Light on Sterol Biosynthesis in Digitalis purpurea L

The effect of varying sequences of red and far red light on sterol biosynthesis in etiolated seedlings of Digitalis purpurea L. was examined. Red light caused a marked increase in the amounts of free and glycosidic sterols and a small decrease in esterified sterols during the first 4 hours after illumination. Far red light elicited the same response but to a lesser degree. Exposure to red followed by far red light or the reverse caused little or no increase in the amounts of free and glycosidic sterols. The magnitude of the increase in the amounts of sterols varied, depending upon the season in which the experiments were performed. The largest increments were obtained during the summer and fall, whereas the smallest were observed during the winter and spring. Correlation of these data with previous observations of an annual cycle in the sterol content of Digitalis seedlings showed that the maximum stimulation in sterol biosynthesis occurs when the endogenous level of sterols is minimal.

Sterol monoglycosides, acylmonoglycosides, and an unidentified sterol conjugate from the lipid extracts were quantitated. Changes in conjugated sterol content were related to the particular light conditions of each experiment. The results are discussed in terms of physiological cycles and the possible influence of hormones upon the control of sterol biosynthesis in Digitalis.

     

Glyceraldehyde 3-Phosphate Dehydrogenases and Glyoxylate Reductase: I. Their Regulation Under Continuous Red and Far Red Light in the Cotyledons of Sinapis alba L

The development of NADP- and NAD-dependent glyceraldehyde 3-phosphate dehydrogenase and NADH-specific glyoxylate reductase was followed in Sinapis alba cotyledons grown in the dark or under continuous red and far red light. All three enzyme activities are promoted by light, continuous far red light being more than twice as effective as continuous red light. The activities of the NADP-glyceraldehyde 3-phosphate dehydrogenase and glyoxylate reductase increase in the far red light from 36 to 96 hours. They remain constant until at least 120 hours after sowing and are respectively 11 and 6 times higher than the maximum dark activities. Contrary to this, the activity of the NAD-glyceraldehyde 3-phosphate dehydrogenase is scarcely more than doubled under continuous far red irradiation relative to its maximal dark level, and its time course curve is displaced along the time axis, with the activity increasing between 24 and 72 hours after sowing.     

Comparison of Three Phytochrome-mediated Processes in the Hypocotyl of Mustard

Anthocyanin synthesis, hair formation, and the synthesis of ascorbic acid oxidase are all phytochrome-mediated reactions occurring in the hypocotyl of mustard (Sinapis alba L.), controlled by phytochrome actually located in the hypocotyl. A comparison of these three reactions showed that in certain respects they differ greatly in their response to light. The ability of the seedling to respond to light by showing the three responses was strongly influenced by the state of development of the seedling. White light given very early after seed imbibition was unable to evoke any of the three reactions. By 50 hours after imbibition, all systems were fully inducible by light. The addition of actinomycin D to a fully competent seedling coincident with illumination strongly inhibited the development of all three responses. In contrast, the addition of cordycepin at this time inhibited the synthesis of anthocyanin and ascorbic acid oxidase but had no effect on hair formation. Cycloheximide inhibited all three responses when given up to several hours after light. This suggests the necessity for RNA and protein synthesis for light-induced expression of these reactions, and that the RNA species involved in the three reactions may have differing degrees of polyadenylation. The lag period between the onset of light and the first display of the response was 3 hours for anthocyanin and ascorbic acid oxidase synthesis, and about 5 hours for hair formation. Amounts of light sufficient to give large increases in the levels of ascorbic acid oxidase and hair formation gave a much smaller increase in anthocyanin synthesis. Hair formation and ascorbic acid oxidase synthesis showed a much greater sensitivity to induction at early stages of seedling development than did anthocyanin synthesis. Following an inductive light period, anthocyanin synthesis was sensitive to far red light inhibition for a period twice as long as the other two reactions. The differences in the response of the three reactions to light suggest that the phytochrome-mediated reactions which control their development also differ.     

Phytochrome-mediated Electric Potential Changes in Oat Seedlings

Brief exposures to red light induce far red-reversible changes of 5 to 10 millivolts magnitude in the upper 1 centimeter of etiolated Avena coleoptiles. The changes begin within 15 seconds of the start of illumination and they continue for at least 12 minutes. The changes were measured using a flowing solution of 10 mm KCl to contact the surface of the coleoptile. A dark-grown coleoptile shows no change in response to far red light unless it first receives red light treatment. The second of two red light exposures is ineffective without an intervening far red treatment. Some characterization of these electric responses to light is presented.     

Phytochrome-mediated Germination Responses in gamma-Irradiated Lettuce Seeds

Sublethal doses of γ-radiation and far red light have some-what analogous, red light reversible, effects on the germination of lettuce seeds (Lactuca sativa L. var. Grand Rapids). However, the mechanism by which γ-radiation retards germination appears to differ from that of far red light. Compared to controls, γ-radiation retarded germination for the first 24 hours; but after 36 or 48 hours of imbibition gemination of treated seeds was higher than that of the controls, whether or not the γ-irradiated seeds received red or far red light. The effects of γ-radiation are more pronounced in seeds containing 15% water at the time of treatment than in those containing only 7% water. The promotive action of red light is operative in the presumed absence of cell division in γ-treated seeds.     

Relation of Phytochrome-enhanced Geotropic Sensitivity to Ethylene Production

Brief exposure of etiolated pea (Pisum sativum cv. Alaska) seedlings to red light enhances subsequent development of geotropic curvature of the stem. Both this response and inhibition of ethylene production by red light become maximal 8 hours after illumination. Very low concentrations of applied ethylene inhibit development of geotropic curvature, whereas hypobaric treatment enhances geotropic sensitivity by removing endogenous ethylene. Increased geotropic sensitivity after illumination is accompanied by increased lateral migration of ³H-indoleacetic acid in response to gravity, and ethylene inhibits this lateral migration. It is suggested, therefore, that red light-enhanced geotropic sensitivity is caused by increased lateral auxin transport resulting from a reduction in ethylene production after illumination.     

Effects of red, far red, and blue light on enhancement of nitrate reductase activity and on nitrate uptake in etiolated rice seedlings

The effects of red (R), far red (FR), or blue light (B) on the enhancement of nitrate reductase (NR) activity and on nitrate uptake in etiolated rice seedlings were examined. On 5-minute illumination followed by 12-hour dark, R caused marked increase of NR activity, but FR and B caused only slight increase. Illumination with 560 ergs per square centimeter per second of R for 5 minutes caused maximal increase. The effect of R was almost completely counteracted by subsequent illumination with 2,000 ergs per square centimeter per second of FR for 10 minutes, indicating that NR induction was mediated by phytochrome. Exogenous supply of inducer nitrate was not required during the 5-minute illumination and the R-FR cycles, if the seedlings were transferred to nitrate solution at the beginning of the dark incubation. NR activity in the shoots was found high when shoots were illuminated but was low when only roots were illuminated. On continuous illumination for 12 hours, B had more effect on NR increase than R.     

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.     

Greening of etiolated bean leaves in far red light

Eight-day-old dark-grown bean leaves were greened by prolonged irradiation with far red light. Growth, chlorophyll content, oxygen-evolving capacity, photophosphorylation capacity, chloroplast structure (by electron microscopy), and in vivo forms of chlorophyll (by low temperature absorption and derivative spectroscopy on intact leaves) were followed during the greening process. Chlorophyll a accumulated slowly but continuously during the 7 days of the experiment (each day consisted of 12 hours of far red light and 12 hours of darkness). Chlorophyll b was not detected until the 5th day. The capacity for oxygen evolution and photophosphorylation began at about the 2nd day. Electron microscopy showed little formation of grana during the 7 days but rather unfused stacks of primary thylakoids. The thylakoids would fuse to give grana if the leaves were placed subsequently in white light. The low temperature spectroscopy of intact leaves showed that the chlorophyll a was differentiated into three forms with absorption maxima near 670, 677, and 683 nanometers at −196 C during the first few hours and that these forms accumulated throughout the greening process. Small amounts of two longer wavelength forms with maxima near 690 and 698 nanometers appeared at about the same time as photosynthetic activity.     

Phytochrome Controlled RNA Changes in Terminal Buds of Dark Grown Peas (Pisum sativum cv. Alaska)

The terminal buds of intact dark grown Alaska pea plants (Pisum sativum) respond to low intensity red light by an enhanced rate of expansion of their leaflets. Twenty four hours after irradiation, red light was found to have increased the ribosomal fraction of the RNA content of the terminal buds. The increase in total RNA liter did not occur if the red light was immediately followed by irradiance with low intensity far red light.