Far-red Light Treatment Research Articles

Photoregulation of β-Tubulin mRNA Abundance in Etiolated Oat and Barley Seedlings

The effect of light on the abundance of beta-tubulin mRNA was measured in etiolated Avena sativa L. and Hordeum vulgare L. seedlings. Slot blot analysis employing an oat beta-tubulin cDNA clone was used to measure beta-tubulin mRNA levels. White light induced a 45% decrease in oat beta-tubulin mRNA abundance by 2 hours after transfer. A saturating red light pulse induced 40 and 55% decreases in beta-tubulin mRNA levels in oats and barley, respectively. Recovery of beta-tubulin mRNA levels was observed after a red light pulse but not after transfer to continuous white light. The red light induced decrease in oat beta-tubulin mRNA abundance was not reversible by a subsequent far-red light treatment. The mesocotyl portion of etiolated oat seedlings exhibited a more dramatic decrease in beta-tubulin mRNA abundance in response to red light than did the coleoptile portion. The results indicate that the well-documented effects of red light on the growth of etiolated seedlings are accompanied by changes in the expression of the beta-tubulin genes.

Response of light-grown wild-type and aurea-mutant tomato plants to end-of-day far-red light

The effect of end-of-day (EOD) far-red (FR) light treatment on tomato ( Lycopersicon esculentum Mill.) plants of the wild type (WT) and an aurea ( au) mutant (which is deficient in the spectrophotometrically detectable light-labile phytochrome pool in etiolated seedlings) was studied. Both the WT and au mutant exhibit a quantitatively similar EOD-FR response ( i.e. stimulation of elongation growth) indicating that phytochrome is functional in light-grown seedlings of the au mutant. However, no dramatic effects of EOD-FR light on leaf elongation, chlorophyll, carotenoid and soluble protein levels were observed in either the WT or au mutant. The au mutant contained less chlorophyll and more soluble protein per unit fresh weight than the WT. Although practically no anthocyanin could be detected in leaves of the au mutant, the WT showed a tenfold reduction in anthocyanin in response to EOD-FR light. The lower productivity of the au mutant compared with the WT results from its reduced leaf surface area, since its maximum rate of photosynthesis or photosynthetic efficiency appears to be slightly higher than that of the WT. Phytochrome was extracted and partially purified from light-grown plants of the WT and au mutant and was quantified by spectrophotometry. On a fresh weight basis the phytochrome content of au-mutant plants was 66% of that of WT plants. It is proposed that the light-labile phytochrome pool regulates the synthesis of the photosynthetic apparatus in light-grown plants and that the light-stable phytochrome pool functions in the EOD-FR elongation response, the latter pool being present and functional in the au mutant.

Phytochrome Mediated Photoregulation of NAD Kinase in Terminal Buds of Pea Seedlings

Summary Exposure of etiolated pea ( Pisum sativum L.) seedlings for 5 min to red light increased NAD kinase activity in the terminal buds after a lag of about 12 h. Maximal increase occurred at 48 h and the activity returned to control level at 96 h of dark incubation. The red-light-mediated increase was reversed by immediate exposure to far-red light demonstrating the involvement of phytochrome in the regulation of NAD kinase by light. The escape period for far-red reversibility was 3 min. Repeated red light exposures given every 24 h caused a much higher increase, which was also reversed by immediate far-red light treatment. Calmodulin levels remain unaltered in seedlings treated with different lights. The phytochromemediated increase in NAD kinase was due to increased synthesis of the enzyme since the effect was abolished when the seedlings were sprayed with actinomycin D and cycloheximide immediately after red light treatment.

Regulation of Glutamine Synthetase by Phytochrome in Pea Terminal Buds

Exposure of etiolated Pea (Pisurn sativum L.) seedlings for 5 min to red light increased glutamine synthetase (GS) activity after a lag of about 8 h. Maximal increase occurred at 30 h and the activity returned to the dark level at 48 h of dark incubation. The red-light mediated increase was reversed by immediate far-red light treatment for 5 min, demonstrating the involvement of phytochrome in the regulation of GS by light. The escape period for far-red reversibility was 2 min. Repeated red light exposures given every 24 h caused a much higher increase which was also reversed by immediate far-red light treatment. Phyto-chrome-mediated increase in GS was abolished if the seedlings were sprayed with actinomycin D and cy-cloheximide immediately after red light treatment. Chloramphenicol had no effect.

The Effect of Light and Phytochrome on 1-Aminocyclopropane-1-Carboxylic Acid Metabolism in Etiolated Wheat Seedling Leaves

While light-grown wheat leaves produced ethylene at a low rate of <0.1 nanomoles per gram per hour and contained 1-aminocyclopropane-1-carboxylic acid (ACC) at low levels of <2.5 nanomoles per gram, etiolated wheat leaves produced ethylene at a rate of 2 nanomoles per gram per hour and accumulated concentrations of ACC at levels of 40 nanomoles per gram. Upon illumination of 8-day-old etiolated wheat seedlings with white light, the ethylene production rate increased initially, due to the activation of ethylene-forming activity, but subsequently declined to a low level (0.1 nanomoles per gram per hour) at the end of the 6-hour illumination. This light-induced decline in ethylene production rate resulted from a decline (more than 35 nanomoles per gram) in ACC level, which was accompanied by a corresponding increase in 1-(malonylamino)cyclopropane-1-carboxylic acid content. These data indicate that illumination promoted ACC malonylation, resulting in reduced ACC level and consequently reduced ethylene production. However, light did not cause any significant increase in the extractable ACC-malonyltransferase activity. The effect of continuous white light on promotion of ACC malonylation was also observed in intermittent white light or red light. A far-red light treatment following red light partially reversed the red light effect, indicating that phytochrome participates in the promotion of ACC malonylation.

Interaction of photoperiod, light intensity, light quality and ancymidol on growth and flowering of Clerodendrum thomsoniae Balf. ‘Wisconsin’

Plants of Clerodendrum ‘Wisconsin’, untreated and soil-drenched with 0.9 mg of ancymidol (cyclopropyl (methoxyphenyl) 5-primidinemethanol), were grown under light intensities of 1492.09 μE s −1 m −2 (20% averaged at mid-day) and photoperiods of 9 h (SD), 16 h (LD) and a 9-h SD with 2 h incandescent light interruption (NI) during the dark period. Plants treated with ancymidol were 1 4 the height of untreated ones. The number of days to flower was least under SD, regardless of light intensity, and greatest under NI at 20% light intensity. Untreated plants produced 18.2, 10.2 and 1.4 flowers per plant under SD, LD and NI, respectively, and ancymidol-treated plants produced 63.6, 62.5 and 34.8 flowers per plant under SD, LD and NI, respectively. Untreated plants grown under natural daylength supplemented with incandescent light to give 16-h days or longer were approximately 7 times taller than treated ones; ancymidol-treated plants averaged 56.0 flowers per plant and untreated ones 0.3. Flower number was greatest in the natural 12-h and least in the 24-h photoperiod. Ancymidol-treated plants exposed to 1 h of red light before the dark period for 2 weeks averaged 48.5 flowers in SD and 55.8 in LD; untreated plants averaged 11.5 and 11.3 flowers, respectively. With far-red light treatment, ancymidol-treated plants produced only a few flowers, and untreated ones remained vegetative in both photoperiods.

Phytochrome-mediated accumulation of chloroplast DNA in pea leaves

Pea seedlings grown for 5 days in the dark were treated with red light for 5 min and grown for 2 more days in the dark. Effects of the red light on chloroplast DNA levels in the pea leaves were examined using probe DNA of the chloroplast-coded large subunit and nuclear-coded small subunit of ribulosebisphosphate carboxylase/oxygenase. The gene dosage of the large subunit, but not of the small subunit, was increased by red light. The increase was inhibited by subsequent far-red light treatment. These results indicate that accumulation of chloroplast DNA in the cell is mediated by phytochrome. Probably the replication of chloroplast DNA is mediated by phytochrome.

Development of cytosol and chloroplast aldolases during germination of spinach seeds

The total activity of aldolase (EC 4.1.2.13) and the activities of cytosol and chloroplast aldolase were determined in seeds, cotyledons, primary leaves and secondary leaves of spinach (Spinacia oleracea L., cv. Monopa) during germination. Total aldolase activity in cotyledons increased from low levels to a low maximum in the dark after one week and to a high maximum in white light after three to four weeks and declined thereafter. The activity in primary and secondary leaves started to rise strongly from the 18th and 26th days, respectively, up to the 42nd day of germination. The levels of aldolase activity paralleled the development of leaf area, chlorophyll content and protein content per leaf except that the leaf area of cotyledons continued to increase steadily up to the 42nd day after the maximum of aldolase activity was reached. Resolution of cytosol- and chloroplast-specific isoenzymes by chromatography on diethylaminoethylcellulose indicated that in the light the cytosol enzyme represented approx. 8% of the total activity in cotyledons, primary and secondary leaves throughout germination, and the chloroplast enzyme represented the remaining 92%. Only in cotyledons of dark-grown seedlings was the cytosol aldolase between 25 and 50% of the total activity. Seeds contained almost exclusively a cytosol aldolase. In cotyledons the increase of total activity in the light was specifically the consequence of an increase in chloroplast aldolase while the cytosol aldolase was little affected by light. The light effect was mediated by phytochrome as demonstrated by classical induction and reversion experiments with red and far-red light and by continuous far-red light treatment.

Immunofluorescence visualization of phytochrome in Pisum sativum L. epicotyls using monoclonal antibodies

We have investigated the cellular distribution of phytochrome in epicotyls of dark-grown pea (Pisum sativum L.) seedlings using monoclonal antibodies to pea phytochrome. Screening of the eight available antibodies both by an enzymelinked immunosorbent assay (ELISA) and by their ability to visualize phytochrome in situ by immunocytochemical fluorescence demonstrated that: (1) three antibodies work well for immunofluorescence; (2) none of the eight antibodies discriminates between the red- and the far-red-absorbing forms of phytochrome (Pr, Pfr) as assayed by ELISA; (3) the antigenicity of phytochrome is reduced by fixation with formaldehyde with respect to all eight antibodies; and (4) two antibodies that bind well to formaldehyde-fixed phytochrome as assayed by ELISA do not bind well to phytochrome in situ. Phytochrome is observed in both cortical and stomatal guard cells of the epicotyl and exhibits a homogeneous cytoplasmic distribution in non-irradiated tissue. After red-light (R) treatment phytochrome becomes transiently inaccessible to antibodies. If maintained in the Pfr form for 10 min at room temperature before fixation, at least a portion of the phytochrome pool becomes accessible to antibodies and assumes a "sequestered" distribution. Both of these effects are almost entirely either prevented or reversed by subsequent far-red light treatment. We believe that the transient inaccessibility of phytochrome to antibodies after R irradiation is not a function of its conformational state. We suggest instead that R treatment rapidly induces an association of phytochrome with a subcellular component that interferes with antibody binding and that the "sequestered" areas represent a phytochrome pool that is distinct from both the diffusely distributed phytochrome in non-irradiated cells and from that phytochrome which is inaccessible to antibodies immediately after R irradiation.

Phytochrome-mediated regulation of two mRNAs, encoded by nuclei and chloroplasts of ribulose-1,5-bisphosphate carboxylase/oxygenase.

Etiolated pea seedlings (5 days old) were treated with red light for 5 min and grown for 2 days more in darkness. Effects of the red light on the mRNA levels of two subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) (ribulose-P2 carboxylase), rate of enzyme synthesis and accumulation of the enzyme were examined for the plants. Increases of the two mRNA levels, encoded in nuclei and chloroplasts were observed. Red light followed by far-red light treatment for 5 min inhibited the increases. An increase parallel with the mRNA levels was found for the rate of the enzyme synthesis and accumulation of the enzyme. These results indicate that the induction of two mRNA levels is mediated by phytochrome and causes the red-light-inducible, far-red-light-reversible increase of ribulose-P2 carboxylase. When white light was irradiated to induce the enzyme, the effects of red light given in darkness were reinforced. These results indicate that light-induction of ribulose-P2 carboxylase is mediated by phytochrome and controlled by the two mRNA levels.

Seed germination in Citrullus lanatus. Part 2. The involvement of phytochrome and ethylene in controlling light sensitivity

The inhibitory effect of light on the germination of Citrullus lanatus seed shows the typical red — far-red photo-reversibility of a phytochrome mediated system. During the dark period following an inhibitory far-red light treatment a secondary skotodormancy develops. The inhibiting effect of light on germination can be effectively reversed by ethylene. Die remmende invloed van lig op die saadkieming van Citrullus lanatus toon die tipiese rooi — verrooillg-omkeerhaarheid van ’n fitochroom-beheerde sisteem. Gedurende die donkerperiode na ’n remmende verrooiligbehandeling ontwikkel daar ’n sekondêre rustoestand by die sade. Die remmende invloed van lig kan doeltreffend deur etileen opgehef word.

ANTAGONISTIC EFFECT OF CPTA AND FAR-RED LIGHT ON THE CAROTENOGENESIS IN LUTESCENT TOMATOES

Yellow lutescent tomatoes normally devoid of lycopene can be induced to produce this red pigment when treated with bioregulators such as CPTA, (2-(4-chlorophenylthio)-triethylamine hydrochloride). Red light and CPTA combination stimulated the accumulation of 225 μg/g of lycopene in mature white lutescent tomatoes after a 12-day ripening period. Tomatoes treated with CPTA but ripened in the dark produced 67 μg/g of lycopene. Far-red light treatment of the CPTA treated tomatoes produced only 25 μg/g of lycopene. Analysis of other carotenoids showed a similar trend indicating a quantitative but not a qualitative change in carotenogenesis in lutescent tomatoes as affected by light and by bioregulators. The antagonistic effect of far-red light toward CPTA suggested that the two effectors might work at the same reactive site in tomato cells.

Hook opening in cucumber seedlings by narrow-band red and far-red light

Intermittent red (R, 660 nm, 3.9 Wm −2) and far-red (FR, 728 nm, 7.0 Wm −2) light treatments caused hook opening in etiolated cucumber ( Cucumis sativus L.) seedlings, by cell elongation at the concave side only, but had no significant effect on hypocotyl elongation. The relative positive effect of FR on the hook increased with age of the seedling. FR counteracted the hook opening effect of R only if given simultaneously with R in young seedlings, whereas FR by itself had no effect or a slightly negative one. Results are not readily explained in terms of high energy response (HER); alternative mechanisms are discussed.

Regulation of Membrane Properties of Mitochondria and Plastids During Chloroplast Development I. The Action of Phytochrome in situ

Summary Mitochondria and plastids were isolated from etiolated laminae of Avena sativa L. given a special light program (different red and far-red light treatments). Both types of organelles exhibited the same changes in the penetrability * of their transport limiting membranes described earlier for white light induced plastid development. Continuous irradiation with far-red light increased the penetrability significantly up to 3 h of light treatment; there-after a decrease in the internal concentration of the 14C-labelled compounds tested (δ-aminolevulinate, succinate) was observed. The same effects were induced after a 5 min treatment with red light followed by darkness. Additionally, induction-reversion experiments performed by irradiating the etiolated seedlings with far-red light (5 min), immediately following the red light irradiations, demonstrated that the effect of red light could be reversed for both mitochondria and etioplasts. Hence, the operational criterion for the involvement of phytochrome is fulfilled. The results therefore indicate that the changes of membrane penetrability observed are mediated by phytochrome.

Floral Induction and the Effect of Red and Far-red Preillumination on the Light-Stimulated Bioelectric Response of Spinach Leaves

Summary It is found that under short day conditions, a low far-red bioelectric response of spinach leaves follows a daily rythm, increasing to a maximum at the end of the day and decreasing in the dark. The response is quickly reversible modified by red and far-red light treatments, and therefore appears to be associated with the presence of phytochrome (short term effect). The far-red response under short day conditions is strongly increased by acetylcholine; DCMU inhibits the photoreversibility of the response. Under long day conditions, we observe a high far-red bioelectric response partially irreversible (> 50%) that is characteristic of the induced state. Once acquired this response does not disappear if the plant is returned to SD conditions for 5 days. We have the same response in the few old plants which flower under SD conditions and in plants treated for 3 days with gibberellin or with acetylcholine. The LD irreversible response seems, therefore, to be in relation with flowering in spinach (long term effect). A spectral index can be used to determine immediately wether a leaf of Spinacia oleracea or Perilla nankinensis is induced. We propose the utilization by the plant of the specific-wavelength stimulated biopotential as electrical signals in coordinating the floral induction.

Developmental studies on microbodies in wheat leaves

1. In etiolated wheat (Triticum aestivum L.) leaves, the development of the microbody enzymes catalase, hydroxypyruvate reductase, and glycolate oxidase was specifically stimulated by short treatments of the seedlings with red light, although the increases were less than observed after treatment with continuous white light. A comparison of the effects of short red and far-red exposures indicated the involvement of phytochrome. 2. Continuous far-red light treatments also enhanced the development of microbody enzymes. Catalase activity continued to increase at a high rate even after return from a prolonged far-red illumination to darkness, while the increase in the activities of glycolate oxidase and hydroxypyruvate reductase fell to the dark rates when the tissue was removed from the light. However, even at higher intensities of continuous far-red light the microbody enzymes reached only considerably lower activities than in white light. During continuous irradiation of equal quantum flux, the microbody enzymes reached higher activities in red than in far-red light, but the highest activities were observed in blue light, which had similar effects as white light. The quantitative difference between the effects of prolonged red or blue light depended also on the seed material and growing conditions. In the presence of the herbicide 3-amino-1,2,4-triazole the increase of glycolate-oxidase activity was reduced in red light but was affected much less, if at all, in blue light. 3. Continuous irradiations with all three light qualities used (red, far-red, blue) influenced the properties of the microbody particles to form a distinct band sharply confined close to an equilibrium density of 1.25 g cm(-3) on sucrose gradients which was not observed in preparations from plant material raised in complete darkness. In preparations from all light-grown plants a special peak in the activity profile of malate dehydrogenase was found in the microbody fraction while it was lacking on gradients from dark-grown leaves. The heights of the activities of malate dehydrogenase as well as of the other enzymes found in the microbody fractions from plants grown in either far-red, red, or blue light differed in the same way as did the activities from total leaf homogenates. 4. Glycolate oxidation by segments of intact leaf tissue was higher with tissue from light- than from dark-grown plants, but after light treatments of different spectral quality its magnitude did not correspond to the extractable activities of glycolate oxidase.

The influence of continuous far-red and white light on prenyl chain synthesis in plastids of Raphanus seedlings

The rate of prenyl chain accumulation (C40 carotenoids; C45 in plastoquinone-9; C20 phytyl in chlorophylls, α-tocopherol and vitamin K1) in plastids of etiolated radish seedlings (Raphanus sativus L.) is determined in continuous darkness and after far-red and white light treatment. Continuous far-red light (active phytochrome P fr ) stimulates the synthesis of all prenyl chains, but has no or only little effect on the dark pattern of the prenyl chain formation. White light enhances the accumulation of prenyl chains to a much higher degree than does far-red light. By a particularly strong promotion of the accumulation of phytyl chains, which are incorporated into chlorophyll, white light changes the percentage composition of prenyl chains to that of chloroplasts.

Potassium flux and leaf movement in Samanea saman. II. Phytochrome controlled movement.

Phytochrome, a membrane-localized biliprotein whose conformation is shifted reversibly by brief red or far-red light treatments, interacts with the rhythmic oscillator to regulate leaflet movement and potassium flux in pulvinal motor cells of Samanea. Darkened pinnae exposed briefly to red light (high P(fr) level) have less potassium in motor cells in the extensor region, more potassium in motor cells in the flexor region, and smaller angles than those exposed to far-red light (low P(fr) level). Increase in temperature from 24 degrees to 37 degrees increases the differential effect of the light treatments during opening (the energetic phase) but not during closure, implying that phytochrome controls an energetic process. It seems likely that phytochrome interacts with rhythmically controlled potassium pumps in flexor and extensor cells. During nyctinastic closure of white-illuminated pinnae, exposure to far-red light before darkening results in larger angles than does exposure to red. As in rhythmic opening, the angles of all pinnae and the differential effect of the light treatments increases with increasing temperature.

Independence of phytochrome destruction and physiological response in Avena and Pisum segment elongation

Control over elongation in Avena coleoptile and Pisum epicotyl segments by cyclic far-red light treatment is dependent on the timing of successive irradiations and independent of total energy. The effect of each far-red irradiation in a cycle is cumulative. Maximum efficiency of far-red light in a cycle is obtained when the light treatments follow within 2 to 3 h of each other. Additional increments of elongation may also be obtained by successive red light treatment only when presented at least 8 to 9 h apart. The effect of a single far-red treatment is to establish a maximum rate of elongation which is maintained for no more than 3 h. The results offer support for the hypothesis that low-energy phytochrome mediated responses result from maintenance of low but maximally effective levels of Pfr and that Pfr destruction is essentially independent of physiological action.

Studies of Achene Dormancy in Fumitory

Fumitory (Fumaria officinalis L.) achenes were subjected to various treatments to induce germination and to study dormancy. Complete pericarp removal, partial seed coat removal, puncturing the seed coat, heat treatment, red and far-red light treatments, and germination temperature treatments did not break dormancy. Low-temperature after-ripening for 60 days at 4 C dormant fumitory achenes resulted in 90 to 95% germination. Water leaching of fumitory achenes increased the time required for successful after-ripening and the water leachate stimulated Avena coleoptile cylinder elongation. Wild oat (Avena fatua L.) embryo germination was inhibited by high concentrations of fumitory achene leachate and stimulated slightly by lower concentrations. High concentrations of the leachate inhibited epicotyl and radicle growth of pre-sprouted lettuce (Lactuca sativa L.) seedlings and lower concentrations markedly stimulated radicle growth.