THE EFFECT OF ORGANIC NITRATES IN PHYTOCHROME-CONTROLLED GERMINATION OF Paulownia tomentosa SEEDS

The long light irradiation requirement in Paulownia tomentosa (empress tree) seeds can be substituted by organic nitrates such as nitroglycerine, isosorbide di- and mononitrate, and pentaerythri-tyl tetranitrate and a pulse of red light (5 min). The most effective was nitroglycerine (0.1 mM). Its effect depended on the time of application, i.e. a simultaneous presence of P_fr and these compounds was required. The effect decreased with delayed time of application after red light pulse. In seeds imbibed in nitroglycerine, an escape from far-red light reversible action was similar to that obtained for seeds which can be induced to germinate by a brief exposure to red light. However, the application of nitroglycerine to seeds after a far-red light pulse was ineffective. The effectiveness of organic nitrates also depended on the number of nitro groups in the compound. Isosorbide mononitrate was less effective than isosorbide dinitrate. Substances with structures similar to nitroglycerine, such as glycerol and glyceryl triacetate, in combination with the pulse of red light, failed to reduce the long light requirement.     

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.     

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.     

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.     

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     

RED-LIGHT-INDUCED GERMINATION OF LETTUCE SEEDS: RELATIONSHIP BETWEEN ESCAPE FROM REVERSIBILITY AND FLUENCE*

Abstract— Dormant seeds of Lactuca saliva L. (cv. ‘May Queen’) germinate after a brief light treatment. The seeds escape from photoreversibility by far-red irradiation after a lag time. The duration of the lag decreases with increasing levels of the far-red absorbing form of phytochrome (P_fr). During the lag time the percentage of seeds germinating after the reversing far-red irradiation is slowly rising. This is not due to an escape reaction proper, but to an increase of sensitivity to P_fr.     

PROBIT ANALYSIS OF LOW AND VERY-LOW FLUENCE-RESPONSES OF PHYTOCHROME-CONTROLLED Kalanchoe blossfeldiana SEED GERMINATION*

Abstract— Kalanchoë blossfeldiana seeds are light-requiring for seed germination. On water or KNO₃ solution and irradiated with several daily red (R) irradiations, the seeds show a low-fluence (LF) response which is far-red (FR) reversible. Incubated on gibberellic acid (GA₃) the seeds show a very-low-fluence (VLF) response which can be saturated with red as well as with far red light. As germination is a quantal response, the sub-optimal segments of the dose-response curves are analysed by means of probit analysis in order to calculate the seed population parameters. There is a linear relation between the probit of the germination response and the logarithm of the fluence. Moreover, the slope for the VLF as well as for the LF response is the same. The VLF requires about 8 × 10⁴ times less fluence than the LF. VLF saturation with FR requires about 200 times more fluence than with R. Although, GA₃ and KNO₃ modulate VLF and LF, respectively, there is no direct influence on the phytochrome-phototransformations. Once P_fr is formed (in VLF or LF, or preserved in dry seeds) germination is proportional to the GA_S concentration (for VLF and dark germination) or proportional to the KNO, concentration (for LF). The non-photochemical events leading to germination seem to be triggered by a similar action mechanism for both GA, and KNO₃.     

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.     

FLUENCE RATE COMPENSATION OF THE PERCEPTION OF RED: FAR-RED RATIO BY PHYTOCHROME IN LIGHT-GROWN SEEDLINGS*

Abstract— The hypothesis that phytochrome functions as a sensor of vegetational shade through the perception of the red: far-red photon fluence rate ratio requires that the mechanism of perception be compensated for wavelength-independent fluctuations in fluence rate (Smith, 1982). This paper seeks to establish the lower limit of fluence-rate compensation and to assess whether or not compensation is effective at the total fluence rates typical of herbaceous canopies. Using specially-designed cabinets, Sinapis alba L. (white mustard) seedlings were grown from germination under a range of total photosynthetically-active radiation (PAR = 400 to 700 nm) values and a range of red: far-red ratios. The data indicate that fluence-rate compensation is effective above a PAR value of ca. 60 μ.mol m² s'. Pretreating seedlings at high red: far-red ratio and a PAR level of 300 (μmol m₂S_-1for increasing periods of time led to an extension of fluence rate compensation to lower fluence rates. The results are discussed in relation to the photosynthetic competence of the seedlings grown under these conditions.     

PHYTOCHROME-MEDIATED RAPID CHANGES IN THE LEVEL OF PHOSPHOINOSITIDES IN ETIOLATED LEAVES OF Zea mays

The role of the active form of phytochrome in Zea mays on the polyphosphoinositide cycle was studied. As little as 15 s of red irradiation of etiolated leaves immediately increased the level of phosphatidylinositol bisphosphate (PIP₂) 3–6-fold compared to unirradiated leaves. The elevated level of PIP₂ decreased with longer red irradiations up to 5 min, but remained higher than in unirradiated leaves. The level of PIP₂ decreased if red irradiation was followed by far-red irradiation. Far-red alone had no effect. Levels of phosphatidylinositol phosphate (PIP) and phosphatidylinositol did not change significantly. Since red irradiation significantly changed PIP, but not PIP, photocontrol appears to be at the PIP kinase and phospholipase level. In related studies of the effect of light on phospholipids, 5 min of red irradiation induced significant decreases in phosphatidylcholine and phosphatidylethanola-mine.     

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.     

HETEROGENEITY OF THE QUINONE ELECTRON ACCEPTOR SYSTEM IN BACTERIAL REACTION CENTERS

Abstract— In reaction centers from Rhodopseudomonas viridis, biphasicity of the charge recombination kinetics between P⁺, the primary electron donor, and Q_A and Q_B^-, the primary and secondary quinone electron acceptors, respectively, have been analyzed by the flash-induced absorption change technique. We have studied the effect of quinone environment modifications on the ratio of the two phases for the P⁺Q_A- ([A_fast/A_slow]^a) and P⁺Q_B^- ([A_fast/A_slow]^b) charge recombination processes. In reaction centers from Rps. viridis reconstituted in phosphatidylcholine liposomes a notable influence of the nature of the Q_B pocket occupancy was observed on (A_fast/A_slow)^a. This ratio is much affected by the presence of o-phenanthroline compared to reaction centers with an empty Q_B pocket or with terbutryn present. Because o-phenanthroline was proposed to hydrogen bind His^L190, whereas terbutryn does not, we suggest that a His^LI90-Fe-His^M217 (the equivalent to His^LI90 in the Q_A pocket) “wire” may be involved in the existence of the two conformational states associated with the two phases of charge recombination. In chromat-ophores from the T₁ (Ser^L223→ Ala; Arg^L217→ His) and T₄ (Tyr^L222→ Phe) mutants no modification of the (A_fast/A_slow)^a ratio is detected, whereas the (A_fast/A_slow)^b ratios are substantially modified compared to the wild type (WT). In the T₃ mutant (Phe^L216→ Ser; Val^M263→ Phe [4.1 Å apart from Q_A]), (A_fast/A_slow)^a is notably changed compared to the WT. Our data show that any modification in the close protein environment of the quinones and/or of the His^L190 and His^M217 affects the equilibrium between the two reaction center states. This is consistent with the existence of two reaction center states from Rps. viridis, associated with two different conformations of the quinones-histidines-iron system. This “wire” allows both quinone protein pockets to interact over quite long distances.     

EVIDENCE FOR PHYTOCHROME CONTROLLED ENDOMITOSIS AND CELL ELONGATION IN PISUM SATIVUM EPICOTYLS

Elongation and endomitosis were studied in the epicotyl cortex cells of germinating seeds of Pisum sativum cv. Rondo. One min of red light per 24 h is sufficient to fully inhibit endomitosis. Terminal far-red irradiation can reverse the red effect to the level established by far-red light alone. This justifies the conclusion that phytochrome is involved in the regulation of endomitotic DNA replication. Since far-red light alone inhibits endomitosis to about 50%, we conclude that very low levels of Pfr are required to influence the endomitotic cycle.     

SIMPLE EXPLANATION OF THE MISSES IN THE COOPERATION OF CHARGES IN PHOTOSYNTHETIC O2 EVOLUTION

Abstract —In the model of Forbush et at. (1971) the observed damping of the flash yield sequence of photosynthetic O₂ evolution was related to a certain percentage of ‘misses’ (α; i.e. centers not converted). The possibility of a miss was supposed to be equal for all states S_0.1,2,3. We propose a new model and a new recurrence law that gives better quantitative agreement with the O₂ yield oscillations observed in Chlorella during a sequence of flashes. We find a better fit with all experimental results by assuming very unequal misses; the misses occur nearly exclusively on S₂ (and also sometimes on S₃). In the simpler case of only one miss on one state, half of S₂ exists as an inactive form S₂₊^- because it is in apparent equilibrium with pool A. The active form of S₂ is converted to S₃ in a flash and the unchanged inactive form S₂₊^- explains the miss: S ₁ hv→S₂₊^-=S₂hv→S₃ (S₂₊^- is a transition state between S₁ to S₂ associated with Q^-). In the dark, the apparent equilibrium constant K_A between pool A and Q (i.e. S₀, S₁ in the dark) is very large; this explains why there is no miss on these states. In light, the experimental value of K_A between pool A and Q (i.e. S₂, S₃ in the light) is 1, and this explains why the misses are large for states S₂, S₃; i.e., S₂₊^-/S2- 1 and sometimes S₃₊^-/S₃?0–1. This new model predicts that the total number of active states ΣS_i=S₀+S₁+S₂+S₃ is an oscillating function of the flash number. This sum 2S, is also the number of trapping centers for excitons. As fluorescence is proportional to excitons that are not trapped, our model explains why the fluorescence oscillates as a function of the flash number. We find also that the initial rates of O₂ evolution after (n - 1) flashes vs the 02 yield of the n^th flash are not exactly on a straight line, which also favors our model.     

THE EFFECT OF RED AND FAR-RED LIGHT ON NUCLEIC ACID METABOLISM IN RYE SEEDLING SHOOTS

Abstract— The quantitative changes in the levels of RNA and DNA in shoots treated with red and/or far-red light are presented.
The results indicate that red light, but not far-red light, stimulated cell division. The changes in RNA are discussed in relation to the nature of the light treatment.     

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.     

GENETIC AND PHYSIOLOGICAL STUDIES OF THE EFFECT OF LIGHT ON THE DEVELOPMENT OF THE MOSS, PHYSCOMITRELLA PATENS

The germination of Physcomitrella patens spores only occurs when wet spores are exposed to light. Depending on their ripeness, spores require from 44 to 64 h illumination to bring about maximum germination. There is a lag period of about 15 h between the reception of sufficient light to elicit germination before germination can be observed. Wavelengths in the range 640–64080 nm are much more effective in inducing germination than longer or shorter wavelengths, but far-red reversal of red light induction of germination has not been demonstrated. Light also has very marked effects on protonemal and gametophore development. In darkness, only caulonemata are produced, and these grow negatively geotropically. No new gametophores develop but existing gametophores grow negatively geotropically, etiolate and bear only scale leaves. In light, chloronemata, as well as caulonemata are produced, the former grow positively phototropically, while the latter grow at right angles to the direction of light, and neither cell type is sensitive to gravity. In the light, gametophores grow positively phototropically, are indifferent to gravity, produce large leaves and do not etiolate. All these responses to light by protonemata and gametophores are shown by cultures growing in a 23 h dark/l h red light cycle, but if this red light treatment is followed by 15min far-red light, the effect of the red light is reversed, indicating an involvement of phytochrome in the mediation of these responses. Mutants showing abnormal growth in the dark have been isolated, as well as mutants having abnormal phototropic responses. The latter type has lost the phototropic response of both the protonemal cell types, as well as of gametophores, indicating that these different responses may share a common component.     

THE EFFECT OF CYTOKININS ON GROWTH and PIGMENT ACCUMULATION OF RADISH SEEDLINGS (RAPHANUS SATIVUS L.) GROWN IN THE DARK and AT DIFFERENT LIGHT QUANTA FLUENCE RATES*

Abstract— The dependency of cytokinin effects upon irradiance was studied with radish seedlings ( Raphanus sativus L. cv. Saxa Treib). Kinetin (6-furfurylamino-purine) or BAP (6-benzylamino-purine) were applied via the roots of plants growing either in continuous darkness or under high (90 Wm^-2) or low intensity white light (10Wm^-2). Apart from the different development of plants at low and high fluence rates, the following cytokinin effects were found:
(1) Both cytokinins acted in a similar manner on growth characteristics and pigment accumulation at high and low light conditions, BAP being in many cases more effective than kinetin.
(2) When compared with the control, the cytokinins suppressed hypocotyl and root lengthening in the dark and light-grown plants. In darkness they led to increased cotyledon areas, whereas in the light the leaf expansion was suppressed.
(3) In the etiolated and low light grown plants, the anthocyanin content of the hypocotyls was enhanced due to the action of cytokinins, whereas under high light the anthocyanin accumulation was decreased.
(4) In the cotyledons of etiolated plants, more phototransformable protochlorophyll(ide) and more carotenoids were formed when cytokinins were present. In green leaves the carotenoid content was diminished due to the action of cytokinins, particularly in plants grown in strong light. The chlorophyll a/b ratio was increased in the cytokinin-treated plants in most cases.
The results suggest a light dependency of the cytokinin effects. It is believed that the response of a plant towards exogenously applied cytokinins is similar to that with high intensity light.     

FLUORESCENCE OF PHYTOCHROME IN THE CELLS OF DARK-GROWN PLANTS AND ITS CONNECTION WITH THE PHOTOTRANSFORMATIONS OF THE PIGMENT

Abstract Fluorescence of phytochrome is found in the cells of etiolated monocotyledonous and dicotyledonous plants. The red light-absorbing form of phytochrome (P_r) fluoresces at 77 K with a yield 0.3±0.1 and maxima at 672–673 nm and 684–686 nm in the excitation and emission spectra, respectively. The emission is characterized by the sharp temperature dependence of its intensity, its high (～ 40%) polarization, and the violation of the mirror symmetry rule. Connection of the fluorescence with P_r photoreactions is followed in the interval 77–293 K. A P, photoproduct, lumi-R, is fluorescent with maxima at 696 nm and 705 nm in the excitation and emission spectra; the far-red light absorbing form of phytochrome (P_fr) is practically nonfluorescent. Three isochromic emitting P_r species are present differing in their photochemical properties: P_r1 and P_r2 which phototransform irreversibly and reversibly at T 170 K into lumi-R, and lumi-R₂, respectively, and P_r3 which undergoes photoconversion only at T > 240 K. The activation energies of P_r2 and P_r3 photoreactions are evaluated to be 2.9–3.3 kJ/mol and 26 kJ/mol. Complex dynamics of changes of P_r fluorescence and of the extent of its decrease in the photoconversion P_r? P_fr in germinating pea and bean seeds suggests the existence of two P_r pools one of which is incapable of P_r? P_fr phototransformation. Thus, the developed fluorescent method of phytochrome assay and investigation in the cell revealing multiplicity of phytochrome states in vivo proves to be very sensitive (about 1 ng) and informative.