PHYTOCHROME-MEDIATED PHOTOTROPISM AND DIFFERENT DICHROIC ORIENTATION OF Pr AND Pfr IN PROTONEMATA OF THE FERN ADIANTUM CAPILLUS-VENERIS L.

Abstract— Single-celled protonemata of Adiantum capillus-veneris were cultured under continuous red light for 6 days and then in the dark for 15 h. Brief local exposure of a flank (5 times 20 /mi) of the subapical region of a protonema to a microbeam of red light effectively induced a phototropic response toward the irradiated side. The degree of the response was dependent upon the fluence of the red light. Red/far-red reversibility was typically observed in this photoreaction, showing that phytochrome was the photo-receptive pigment. When the flank was irradiated with a microbeam of linearly polarized red and far-red light, red light with an electrical vector parallel to the cell surface was most effective. However, the far-red light effect was most prominent when its electrical vector was normal to the cell surface. These polarized light effects indicate the different dichroic orientation of P_r (red-light-absorbing form of phytochrome) and P_r (far-red-light-absorbing form of phytochrome) at the cell flank.     

PHYTOCHROME-MEDIATED PHOTOTROPISM IN PROTONEMATA OF THE MOSS Ceratodon purpureus BRID

Abstract— Protonemata of the moss Ceratodon purpureus cultured in white light were transferred to darkness for 3 days and then used for phototropic experiments. Irradiation of the apical region of vertically position protonemata with small beams (0.2 mm) of red light induced a growth response towards the irradiated side (positive phototropism). The phototropic response showed irradiance dependence. The effect of red light was completely reversed by far-red light following red light irradiations, demonstrating that phytochrome was the photoreceptor pigment. Far-red light or UV-blue light had no influence on either bulging or phototropism. Experiments with linearly polarized red or far-red light showed a different dichroic distribution of phytochrome in its different forms, the red-absorbing form, P_r and the far-red-absorbing form, P_fr. Red light with a vibration plane parallel to the long axis of the filaments was most effective. The effectiveness of far-red light was expressed best when its vibration plane was 90° to the electrical vector of the inductive red light.     

LIGHT REQUIREMENTS FOR THE ENHANCED SYNTHESIS OF A PLASTID mRNA DURING SPIRODELA GREENING

A plastid mRNA (5 × 10⁵ mol wt) appears as a burst 3 h after white light greening of steady state dark grown plants of Spirodela oligorrhiza. In this species, chlorophyll synthesis begins after 12 h. The light requirement is different from the pulse of far-red reversible red light required to abolish the lag of chlorophyll synthesis in many species, including Spirodela. Continuous high energy far-red is not stimulatory. When the illumination is not continued throughout the time of incorporation, the stimulation is minimal. Low energy blue and red light are stimulatory, and green and far-red light are ineffectual. Blue light was > 5 times as effective as red light at many dose levels. Illumination with 3 × 10¹⁷ quanta/m²/s (50pEm/cm²/s) blue light at 476 nm gave about half maximum stimulation.     

INTRACELLULAR DICHROIC ORIENTATION OF THE BLUE LIGHT-ABSORBING PIGMENT AND THE BLUE-ABSORPTION BAND OF THE RED-ABSORBING FORM OF PHYTOCHROME RESPONSIBLE FOR PHOTOTROPISM OF THE FERN Adiantum PROTONEMATA

The intracellular localization and orientation of the receptors for the blue light-induced phototropism in the fern Adianrum protonemata, phytochrome and the blue light-absorbing pigment, were investigated by combining the techniques of cell centrifugation and of microbeam irradiation with linearly polarized light. The phototropic response was induced in the cells even after they had been centrifuged basipetally to spin down the endoplasm from the apical region. When a polarized blue microbeam was given to a flank of the apical region of the protonema, the phototropic response after compensation of phytochrome effect by far-red light was most effectively induced when the polarization plane was parallel to the long axis of the cell. If protonemata were pre-irradiated with blue and far-red light, the phototropic response was mediated through phytochrome alone. If such pre-irradiated protonemata were similarly irradiated with a polarized blue microbeam, polarized light vibrating parallel to the cell axis was again most effective in inducing the response. These results indicate that both the blue light-absorbing pigment and the phytochrome responsible for the blue light-induced phototropism in Adiantum are confined to the plasma membrane and/or the ectoplasm and that the transition moments of their blue-absorption bands are nearly parallel to the cell surface.     

THE TEMPERATURE DEPENDENCE OF Pfr ACTION GOVERNS THE UPPER TEMPERATURE LIMIT FOR GERMINATION IN LETTUCE

In most cultivars of lettuce (Lactuca saliva L,), red light acting through the red/far-red reversible phytochrome system promotes full germination within the20–30°C range, but at progressively higher temperatures germination declines sharply. The relationship between this upper ternperature limit for germination and the temperature dependence of phytochrome action was investigated in Grand Rapids lettuce. In fresh seeds the GT₅₀ (temperature giving half maximal germination) was ca 29–30°C. In these seeds, escape from far-red reversibility did not occur at 35°C, a temperature above the GT₅₀, but occurred rapidly at 27°C, a temperature below the upper limit. Increasing periods of dark pretreatment at high temperature (35°C) or increasing concentrations of the germination inhibitor coumarin caused a progressive decline in the GT₅₀, Escape from photoreversibility did not occur at 27°C in seeds in which the GT₅₀ had been reduced to less than 25°C by coumarin or by prolonged high temperature pretreatment. These results indicate that there is a close correlation between the position of the upper temperature limit for germination, and the temperature dependence of phytochrome action. We conclude that factors that alter the upper temperature limit for germination do so by changing the temperature dependence of phytochrome action.     

THE CONTROL BY PHYTOCHROME OF NITRATE REDUCTASE IN THE CURD OF LIGHT-GROWN CAULIFLOWER

Abstract— The activity of nitrate reductase from the curd of light-grown cauliflower ( Brassica oleracea (L) var botrytis (DC) 'St. Hilary') is modulated by nitrate and by light. Using broad-band sources of equal photosynthetically active radiation but with different proportions of red and far-red light, a linear relationship between nitrate reductase activity and ψ(Estimated phytochrome photoequilibrium) was obtained. This relationship, apparent after 8 h incubation, was maintained and little altered after 48 h incubation. The linearity was apparent between ψE 0.26 and ψE 0.69; ψE 0.26 being no more effective than a dark control. Far-red reversibility confirmed the involvement of phytochrome. Brief pulses of red light were also used to establish a range of phytochrome photoequilibria within the tissue. Again a linear relationship between ψ and nitrate reductase activity was obtained with a threshold for the response at ψ 0.3. With both monochromatic and broad-band sources it was seen that neither photon fluence rate nor duration of exposure affected the final activity of the enzyme and that phytochrome was acting solely through ψ (or [Pfr] since phytochrome is stable in this tissue) to bring about these responses.     

EFFECTS OF INORGANIC NITROGEN ON THE RESPONSE OF LEMNA CARBON DIOXIDE OUTPUT TO LIGHT QUALITY AND TIMING*

Abstract —The effects of various light/dark schedules on the time course of CO₂ output by axenic cultures of the short-day plant Lemna perpusilla 6746 differ substantially depending on whether the medium is N-less or contains NH₄ or NO₃ as the sole N source. The steady-state pattern achieved with a daily 1/4 h light pulse in N-less medium is essentially the same whether the light is red or far-red; on NO₃ or NH₄, however, the red and far-red patterns differ in form and suggest the action of a ‘Pfr-hourglass’ timer. In darkness, following either continuous light or entrainment to kh red light daily, CO₂ output oscillates for three or more circadian cycles on NH₄ medium and for at least two on N-less, but damps after a single cycle on NO₃. A schedule of 1/4 h red light every 12 h elicits a 24 h periodicity on NO₃ or NH₄ media and a 12 h periodicity on N-less medium, while a similar far-red schedule elicits a 12 h periodicity on all three. CO₂ output patterns on each of the media respond differently to varying the daily span of light from 1/4 to 6 to 12 h. These results are probably due to differential effects of changing N status on the proportion of total C O₂ arising from various metabolic reactions. They suggest that, rather than being a simple, unitary indicator, CO₂ output can be made to reflect different processes on different media, increasing its value as a real-time indicator of events underlying photoperiodism.     

ANALYSIS OF LIGHT-CONTROLLED ANTHOCYANIN FORMATION IN COLEOPTILES OF Zea mays L.: THE ROLE OF UV-B, BLUE, RED AND FAR-RED LIGHT

Abstract— Light-induced anthocyanin formation in Zea mays L. coleoptiles was investigated in seven different varieties of this species. Under the test conditions, four varieties showed practically no response to any waveband used (UV, continuous red and continuous far-red), two responded strongly to both UV and far-red, and one showed a strong response only to far-red. The radiation-sensitive varieties showed, however, only a very weak response to continuous red light. In those varieties sensitive to far-red light, a pretreatment with continuous red light led to a greatly enhanced response to UV or in one case the manifestation of a response to UV that was previously lacking. Further investigations in one radiation-sensitive variety (INRA) showed that the UV response was to UV-B radiation below 350 nm. The UV response, as well as the far-red and blue responses in this variety, showed fluence-rate dependency. Red light was almost ineffective and showed only a very weak fluence-rate dependency.     

CONTROL OF HYPOCOTYL GROWTH IN MUSTARD SEEDLINGS AFTER LIGHT-DARK TRANSITIONS

Abstract— Six hours of irradiation with white or far-red light strongly stimulates the 'end of day' inhibition of hypocotyl elongation in dark grown Sinapis alba L. Measurements of both reversion kinetics of the inductive light pulse and 'null point' experiments (Hillman, 1965. 1972) indicate that this response is controlled by stable phytochrome. The extent of the reversible response (i. e. length after far-red light minus length after red light) showed rhythmic oscillations after a 6, 48 and 54 h white light pretreatment. The rhythm is started by the transition from light to dark and neither phase nor amplitude is influenced by the level of P at this transition.     

LIGHT PROMOTION OF SEED GERMINATION IN Datura ferox IS MEDIATED BY A HIGHLY STABLE POOL OF PHYTOCHROME

Abstract— The duration of the far-red light-absorbing form of phytochrome (Pfr) of the photoreceptor pool involved in the control of seed germination was investigated for Datura ferox seeds. These seeds require both Pfr and alternating temperatures (20/30°C) to germinate. After 24 h imbibition (25°C), the seeds received pretreatment-light pulses providing different phytochrome photoequilibria (Pfr/P), followed by a 24 h dark incubation (25°C), and test-light pulses providing different Pfr/P immediately prior to transfer to alternating temperatures. Germination increased with increasing Pfr/P provided by the test-light pulses, but was unaffected by the pretreatment-light pulses. This suggests that phytochrome synthesis, phytochrome degradation and phytochrome-mediated changes in response to phytochrome were negligible. In other experiments, red light-pretreatment pulses were followed by dark incubations (25°C) of different duration before transfer to alternating temperatures. The proportion of Pfr remaining after the 25°C incubation period was estimated by comparing germination rates with those of seeds that received test-light pulses of known calculated Pfr/P immediately prior to the start of the cycles of alternating temperatures. More than 80% of the Pfr established by a Pfr/P= 0.87 light pulse was present and active even after 48 h dark incubation at 25°C. Surprisingly, when a pretreatmentlight pulse providing a Pfr/P= 0.70 was given, the reduction in [Pfr] was significantly faster.
Germination of Datura ferox seeds is under the control of a highly stable (type II like) phytochrome pool. Apparently, this pool follows Pfr dark reversion to the red light-absorbing form, the times to reach half the original Pfr pool being > 96 h or <14 h after light pulses providing Pfr/P= 0.87 or 0.70, respectively.     

DOSE RESPONSE BEHAVIOUR FOR POLAROTROPISM OF THE CHLORONEMA OF THE FERN DRYOPTERIS FILIX-MAS (L.) SCHOTT

Abstract— The dose response behaviour for polarotropism of the chloronema of the fern Dryopteris filix-mas was studied in detail. By varying the two factors, time and intensity, dose response curves were obtained, which consisted of as many as two maxima and minima or no maxima and minima at all. Furthermore, the effects of red light and dark, given before polarotropic induction, were compared, and the influence of temperature on the dose response behaviour was examined. There are two main results: (1) Under all conditions tested, the dose response behaviour in far-red, red, blue, and near U.V. apparently is the same. (2) In this one system, the chloronema of Dryopteris , all types of dose response curves, which have been described previously for tropic responses mediated by light, could be obtained simply by varying the conditions. The variable and complex dose response behaviour is discussed in connection with other photoresponses in lower and higher plants.     

Gradient formation of anthocyanin in seedlings of Fagopyrum and Sinapis unilaterally exposed to red and far-red light

Abstract— Anthocyanin synthesis was measured in hypocotyl halves excised from Fagopyrum and Sinapis seedlings irradiated unilaterally or equilaterally with red or far-red light. Although no phototropic curvatures are produced by red or far-red exposure, a significant gradient in anthocyanin formation was observed for Fagopyrum seedlings and the trend for gradient formation is present in Sinapis seedlings. The gradient production in Fagopyrurn is correlated with this tissue's greater optical density. Since the intensities used do not inhibit elongation completely (through the phytochrome system) and an intensity gradient is present in the tissue (as evidenced by anthocyanin formation) it is concluded that (a) a steeper light gradient is required to induce phototropic curvatures or (b) a diffusible material affecting elongation growth prevents any differential from being established across the tissue.     

Light Regulation of Phytochrome Content in Wild-type and Aphototropic Mutants of the Moss Ceratodon purpureus

In filaments of the moss Ceratodon purpureus , phototropism is controlled by the photoreceptor phytochrome. Thirty-three aphototropic mutants with a proposed defect in phytochrome-chromophore biosynthesis were isolated and analyzed. The phototropic response of those mutants was rescued with the precursor of the phytochrome chromophore, biliverdin. Phytochrome spectral activity was measured in 19 arbitrarily chosen mutants. All contained low but still measurable quantities of photoactive phytochrome; the highest level was around 15% of the wild-type. The level of total phytochrome (apophytochrome and holophytochrome) as assayed by immunoblotting was indistinguishable from wild-type. The content of photoactive phytochrome in Ceratodon is light-regulated. Phytochrome of wild-type kept for 24 h in red light was reduced to 50% as compared to dark controls but was unaffected by blue. The red-light-induced decrease was partially reversible by far-red light, indicating that phytochrome itself is the photoreceptor for this response. This regulation was further analyzed with the mutant ptr114 , which contains 15% photoactive phytochrome as compared to the wild-type. In this mutant, continuous red light given for 6 days decreased the level of spectrally active phytochrome down to 25% of dark controls, whereas the amount of phytochrome found on immunoblots was hardly reduced. This indicates that the loss of phytochrome affects only the holoprotein and implies that Ceratodon phytochrome is specifically degraded as a far-red-absorbing phytochrome.     

Photoreception and Phototropism in Phycomyces: Antagonistic Interactions between Far-UV, Blue, and Red Light

Abstract— Phototropism of the sporangiophore of the fungus Phycomyces is mediated by UV and blue light. Classical phototropism action spectra with maxima near 280, 370 and 450 nm indicate a flavin-like photoreceptor. Blue light mediates positive phototropism while far-UV light mediates negative phototropism. To better understand the mode of interaction of far-UV with blue light we performed phototropism experiments in which sporangio-phores were placed for 4 h between sources of 280 and 454 nm light coming from opposite directions. The fluence rates of the far-UV were chosen such that unilateral light alone elicited 90° of negative bending. For blue light, moderate fluence rates were applied that elicited about 40° bending. Under conditions of bilateral irradiation the blue light substantially reduced the far-UV elicited phototropism. In the presence of tonic red light the antagonism between far-UV and blue light was greatly reduced. Red light, which by itself is phototropically ineffective, also reduced phototropic bending elicited by either far-UV or blue light. These observations are taken as indications for the existence of a red light-absorbing intermediate of the blue-light receptor. Because the far-UV/ blue-light antagonism disappeared almost completely in the presence of tonic red light, the antagonism may occur at the level of this receptor intermediate.     

MODE OF COACTION OF PHYTOCHROME AND BLUE LIGHT PHOTORECEPTOR IN CONTROL OF HYPOCOTYL ELONGATION

Abstract The rate of hypocotyl longitudinal growth in seedlings of Sesamum indicum L. is strongly inhibited by continuous blue light (cBL)† and slightly by continuous far-red light while continuous red light (cRL) or red light pulses are hardly effective from 60 h after sowing onwards. Between 36 and 60 h after sowing the growth rate responds to red light pulses the effect of which is fully reversible by long wavelength far-red light. When seedlings are kept in cBL for 3 days and then treated with red light hypocotyl growth rate responds strongly. However, RL effectiveness decreases with time after transfer from BL to RL. BL → darkness transfer experiments with different levels of P_fr established at the beginning of darkness show that after a BL pretreatment phytochrome (P_fr) alone is capable of fully controlling growth rate. When white light (WL) is given no BL effect is detectable in weak WL. Only high light fluxes maintain a typical BL growth rate. At medium WL fluxes elongation rate returns gradually to the dark rate. The simplest explanation of the data is that light absorbed by a separate BL photoreceptor is necessary to maintain responsivity to P_fr. With increasing age of the seedlings the requirement for BL increases strongly. On the other hand, brief light pulses—given to demonstrate photoreversibility of phytochrome—remain equally effective provided that responsivity to P_fr exists.     

Phytochrome-mediated photocontrol of the germination of the scentless mayweed, Matricaria inodora L., and its sensitization by nitrate and temperature

The emergence of Matricaria inodora (M. perforata) may poorly respond to modified light exposure during tillage. Therefore, the influence of light, nitrate and temperature on the germination of M. inodora has been studied under laboratory conditions. Germination of a native seed batch levels off around 77% after 7 to 9 days at 20°C, if sown in 0.01 M KNO₃ or NaNO₃ with a daily photoperiod of 14 h weak white light. In 0.01 M NaCl or in bidistilled water germination reaches 48 ± 5%. One saturating red-light pulse, applied 12 h after imbibition in 0.01 M KNO₃, gives only 12 ± 3% germination after 7 days, but 5 daily repeated red-pulses produce 75% germination. Ten twofold daily repeated red-light pulses are equivalently efficient, and if all immediately followed by a saturating far-red pulse, perfect reversibility down to the far-red level of 7 ± 3% germination is found. The dark germination was 1.7 ± 0.7%.

A fluence-response curve, elaborated for 10 red-pulses, gives an exponential pattern with half-saturating pulse fluence around 35 J m⁻², as indicative of phytochrome photoconversion. The influence of additional temperature pretreatment in darkness was tested, followed by half-saturating red-light pulses every 12 or 24 h. Chilling at 2 °C was ineffective up to 6 days, but slightly sensitizing after 21 days, giving around 30% and 46% germination, respectively. Preincubation for 6 days between 20 and 36 °C was more efficient and led to 58–68% germination at 20 °C. A thermoperiodic pretreatment with daily 12 h at 10 °C and 12 h at 30 °C was most efficient, giving 73 ± 4% germination. For all these pretreatments dark germination never exceeded 3%.

Elevated dark germination occurred when water-imbibed achenes, being exposed and dried in red-light of 1 W m⁻², have been reimbibed in darkness. If drying in red-light was for 6 h to a water content of 20% and reimbibition was in water or 0.01 M KNO₃, dark germination increased to 22 ± 5% or 40 ± 6%, respectively. This dark germination was far-red reversible down to 5 ± 2% with half-escape time around 5 h. If drying in red-light was for 24 h to reach the storing content of water around 11%, the dark germination after reimbibition in water or 0.01 M KNO₃ was only 4 ± 2% and 13 ± 2%, respectively. All corresponding controls for drying and reimbibition in darkness, for both water and nitrate, never exceeded 1% germination. This means that drying of seed in light — but not in darkness — increases emergence after the next rainfall and that the nitrate ion acts as a sensitizer for preformed P_fr and facilitates phytochrome-mediated germination.

Thus, for maximum germination of surface exposed M. inodora a thermoperiodic pretreatment, daily repeated light exposures and nitrate are needed. This is typical of the shallow germinating ancestor occurring in coastal ranges, the Sea Mayweed, Tripleurospermum maritimum, and may explain why the emergence of M. inodora is scarcely reduced after lightless tillage.     

PHYTOCHROME AND EFFECTS OF SHADING ON GROWTH OF WOODLAND PLANTS

qrowth of Circaea lutetiaim plants was studied in various locations in or near a mixed deciduous woodland. Morphological changes resulting from increased shading included increases in leaf area ratio, specific leaf area and specific water content. Parallel measurements with a spectroradi-ometer confirmed that shading involved a reduction in both light fluence rate and light quality (e.g. red/far-red ratio). Phytochrome P_fr/P status was also studied by spectrophotometric measurements on Avena seedling test material and by biological (Lactuca seed germination) assay. Attempts were made to demonstrate phytochrome controlled changes in plant morphology under controlled environment, using both end-of-day far-red treatment and far-red enrichment of the main light period. Effects of natural shading were most clearly simulated by varying light fluence rate while maintaining a constant but high red/far-red ratio     

PHYTOCHROME CONTROL OF ITS OWN SYNTHESIS IN Sorghum vulgare AND Avena sativa

The accumulation of phytochrome in the dark was measured for Avena sativa seedlings after a white light pretreatment and for Sorghum vulgare seedlings after continuous red or far-red light treatments, using the herbicide Norflurazon to prevent greening under continuous irradiation. In both cases the accumulation of phytochrome depends on the state of the phytochrome at the light-dark transition: high P_fr levels (red light pulse) led to a slower rate of phytochrome accumulation than lower P_fr levels (long wavelength far-red (RG 9) light pulse). Poly-(A⁺)-RNA was isolated fromA. sativa seedlings grown for 48 h in darkness + 24 h WL + light pulse (5 min) (red, RG 9 light, red followed by RG 9 light or RG 9 followed by red light pulse) + 19 h darkness. The poly-(A⁺)-RNA was translated in a rabbit reticulocyte lysate system and the translation products were immunoprecipitated by specific anti-phytochrome antibodies. It was demonstrated that the activity of mRNA coding for phytochrome was under phytochrome control.     

INTERACTION BETWEEN PHYTOCHROME AND THE BLUE LIGHT PHOTORECEPTOR SYSTEM IN Mougeotia*

Abstract— Face-to-profile chloroplast movement in Mougeotia was induced by sequences of strong blue and red short irradiations. This type of response occured only when blue light was applied prior to or simultaneously with red light, and far-red irradiation was necessary after the sequence to cancel the remaining gradient of the far-red absorbing form of phytochrome P_fr. The dependence of the response magnitude on blue and red light sequences was studied for a wide range of light durations and dark intervals. The relationship between the response and the dark interval points to the lack of direct coupling between phytochrome and blue-absorbing “cryptochrome”. It was postulated that a photoproduct having a life-time of2–3 min is formed by the blue-light-mediated reaction. This photoproduct interacts with phytochrome during its transformation or with its final P_fr form.     

Phototropism: a "simple" physiological response modulated by multiple interacting photosensory-response pathways

Phototropism is the process by which plants reorient growth of various organs, most notably stems, in response to lateral differences in light quantity and/or quality. The ubiquitous nature of the phototropic response in the plant kingdom implies that it provides some adaptive evolutionary advantage. Upon visual inspection it is tempting to surmise that phototropic curvatures result from a relatively simple growth response to a directional stimulus. However, detailed photophysiological, and more recently genetic and molecular, studies have demonstrated that phototropism is in fact regulated by complex interactions among several photosensory systems. At least two receptors, phototropin and a presently unidentified receptor, appear to mediate the primary photoreception of directional blue light cues in dark-grown plants. PhyB may also function as a primary receptor to detect lateral increases in far-red light in neighbor-avoidance responses of light-grown plants. Phytochromes (phyA and phyB at a minimum) also appear to function as secondary receptors to regulate adaptation processes that ultimately modulate the magnitude of curvature induced by primary photoperception. As a result of the interactions of these multiple photosensory systems plants are able to maximize the adaptive advantage of the phototropic response in ever changing light environments.