A TWOFOLD ACTION OF PHYTOCHROME IN CONTROLLING CHLOROPHYLL a ACCUMULATION

Abstract— A pretreatment with light prior to continuous illumination with high intensity white light eliminates the lag phase in chlorophyll a accumulation and increases the steady-state rate of chlorophyll a accumulation. In mustard seedlings ( Sinapis alba L.) the effect of a pretreatment can be fully attributed to phytochrome. The effect of phytochrome on chlorophyll a accumulation is twofold. It is possible to separate the effect on the lag phase from the effect on the steady-state rate of accumulation. While the effect on the lag phase is a relatively fast process (occurring within less than 3 h) the effect on the rate requires a considerable period of time (at least 12 h) to become manifest.     

PHYTOCHROME IN ETIOPLASTS IN RELATION TO CHLOROPHYLL ACCUMULATION*

Abstract— De-etiolation of maize seedlings reduces their sensitivity for red light potentiation of rapid chlorophyll accumulation in white light. An earlier proposal (Raven and Spruit, 1973) attributes this to migration of the far-red absorbing form of phytochrome (P_fr) to receptors essential for chlorophyll synthesis, thereby increasing the local P_fr/total phytochrome (P_tot)ratio. We have studied etioplasts as possible loci for such P_(r receptors. The level of spectrophotometric phytochrome in purified etioplasts isolated from red preirradiated maize seedlings was higher than that of dark grown plants. The difference was marginally significant, however. We argue that migration of a fraction of cytoplasmic P_fr to the etioplasts, too small to be spectrophotometically demonstrable, could still meet the requirements of the model. Dark destruction of bulk spectrophotometric P_fr following saturating red irradiation of seedlings is not paralleled by a decrease of etioplast phytochrome. the latter remaining essentially constant over long periods. On the other hand, the potentiating effect of red light in intact seedlings is still partially reversible by far red light even after 24 h of darkness when destruction of bulk P_fr is complete. Since this demonstrates persistent presence of P_fr active in potentiation, we propose that at least part of this P_fr is associated with the etioplasts.     

CONTROL BY PHYTOCHROME OF CHLOROPHYLL SYNTHESIS IN SEEDLINGS OF SORGHUM VULGARE

The strong effect of light pretreatments on the synthesis of chlorophyll-a and-b in the shoot of Sorghum vulgare (kept under saturating white light) can be attributed to phytochrome only. No specific blue light effect was found. The phytochrome system appears to function perfectly normally under these conditions. Escape from reversibility is not detectable up to approximately 40 min after the onset of an inductive red light pulse. Thereafter, escape is fast, being completed at approximately 2.5 h after the inductive light pulse.     

CONTROL OF CHLOROPHYLL b BIOSYNTHESIS BY PHYTOCHROME

In the mustard seedling cotyledons, chlorophyll b appears from the very beginning in white light provided that a red light pulse pretreatment was given 12 h prior to the onset of white light. The red light pulses act through phytochrome. Without pretreatment no chlorophyll b is detectable at least during the first 60 min after the onset of white light (25°C). Biogenesis of chlorophyll b specifically depends on the action of phytochrome during the pre-steady state period as well as during the steady state period of chlorophyll accumulation. In light pulse experiments, it was found that formation of chlorophyll b takes place stoichiometrically at the cost of chlorophyll(ide) a.     

CHLOROPHYLL METABOLISM IN ETIOLATED GHERKIN SEEDLINGS I. PHOTOINHIBITION OF CHLOROPHYLL ACCUMULATION

Abstract. Cotyledons of etiolated gherkin seedlings do not turn green upon transfer to high intensity red light (about 25 W/m²). A pre-irradiation with high intensity red light has an after-effect as chlorophyll accumulation during a subsequent exposure to white light (20 W/m²) is inhibited.
The capacity of protochlorophyll regeneration during a dark period depends on the length of a previous light period but is hardly affected by the light intensity. At high intensity light the rate of protochlorophyll regeneration, which also depends on the length of the foregoing irradiation, is lower than that at low intensity light only during the first 1.5h of the light period. It is concluded that high intensity red light inhibits chlorophyll accumulation mainly by photo-bleaching of chlorophyll. The after-effect is the result of a photooxidation which may lead to photo-bleaching of newly formed chlorophyll in relatively low intensity light.
Photoinhibition of chlorophyll accumulation is accompanied by a disturbed development of etioplasts into chloroplasts.     

A POSSIBLE CONTROL BY PHYTOCHROME and OTHER PHOTORECEPTORS OF PROTEIN ACCUMULATION IN THE GREEN ALGA Ulva rigida

Abstract— The effects of red, blue and green light pulses on total protein accumulation in the green alga Ulva rigida following transfer from a low to high nitrate medium in darkness were examined. Red light pulses prior to transfer to darkness increased protein accumulation by about 55%. Blue and green light pulses also stimulated protein accumulation, but to a lesser extent (40-30% respectively). Stimulation of protein accumulation by red, blue or green light was largely (red light) or partially (blue or green light) reversible by far-red. The role of phytochrome and a red/green photoreversible system in the control of protein synthesis is discussed.     

PHOTOREGULATION OF LOGARITHMIC FLUENCE-RESPONSE CURVES FOR PHYTOCHROME CONTROL OF CHLOROPHYLL FORMATION IN PISUM SATIVUM L*

Abstract— Logarithmic fluence-response curves for red (660 nm) and far red (730 nm) light induction of rapid chlorophyll a (Chi a) accumulation in pea seedlings (Pisum sativum L. cv. Early Alaska) indicate extreme light sensitivity in dark-grown seedlings. The energy requirement for onset of 660 nm light induction is less than 20 μJ m^-2 and for 730 nm is about 1 mJ m^-2. De-etiolation produced by a saturating exposure of red light (3–8 kJ m^-2) 24 h prior to the construction of the logarithmic fluence-response curves resulted in approximately a 3 fold increase in slope for 660 nm light, whereas the energy requirement for onset of induction shifted to about 100 mJ m^-2. In such de-etiolated plant material, far red applied at low incident energies almost completely lost its inductive capacity. The inductive capacity of far red applied as high irradiance over a long period of time (16h) appeared not to be affected by the de-etiolation treatment. Reciprocity failed for both dark-grown and de-etiolated seedlings upon exposures exceeding 1,000s. Nearly identical results were obtained for seedlings de-etiolated by red exposures immediately followed by far red (4.8 kJ m^-2), although this treatment did not lead to any significant decrease in spectrophotometrically measurable phytochrome. Therefore, no simple correlation was observed between the level of phytochrome present and the sensitivity of seedlings for induction of rapid Chl a accumulation. In order to explain this apparent phytochrome paradox the possibility was tested and ruled out for changes in the degree of synchronization of seedlings, or for induction of some sort of circadian rhythmicity in light sensitivity being involved. In addition, no correlation was observed between induction of morphogenic development and changing light sensitivity. These results formed, therefore, additional support for a model for phytochrome action involving its intracellular transport and local concentration during the process of seedling de-etiolation.     

REGULATION OF PROTEIN PHOSPHORYLATION BY PHYTOCHROME IN Sorghum bicolor

Abstract— In vitro phosphorylation of some polypeptides was affected in extracts obtained from 5-and 6-day-old plants irradiated with 5 min of red light. The phosphorylation of 55 kDa polypeptide in both 5- and 6-day-old plants, a 60 kDa, and 76 kDa polypeptide in 6-day-old plants and 70 kDa, 67 kDa polypeptide in 5-day-old plants was stimulated by red light. This effect was reversible by far-red light. The extent of stimulation by red light and reversal by far-red light varied for different polypeptides. No differential effect of red and far-red light was seen on the phosphorylation of 94 and 40 kDa polypeptides. In fact, phosphorylation of 94 kDa polypeptide in 6-day-old plants decreased on red light irradiation. These results show that the phosphorylation or dephosphorylation of some proteins is affected by phytochrome and the effect of light is also dependent on the age of the plant.     

PHOTOCONTROL OF ANTHOCYANIN SYNTHESIS: PHYTOCHROME,CHLOROPHYLL AND ANTHOCYANIN SYNTHESIS*

Abstract —Anthocyanin synthesis in cabbage and mustard seedlings depends upon duration and irradiance of the light treatment. The relative effectiveness of radiation in various spectral regions depends upon the length of the irradiation and decreases with increasing dose. In intermittent light treatments, far-red light can reverse the promoting action of red light if the dark interval between successive irradiations is longer than one hour. If the length of the dark interval is less than one hour, far-red applied immediately after each red irradiation, enhances anthocyanin accumulation. Anthocyanin accumulation under various light treatments seems to correlate, to some extent, with the rate of phytochrome decay, but not with chlorophyll production. Anthocyanin accumulation is inhibited by 2,4-dinitrophenol and by the ammonium ion, but not by DCMU. The ammonium ion inhibits anthocyanin accumulation induced by a single, short red irradiation. This suggest that the ammonium ion may have a wider spectrum of action in vivo than in chloroplast preparations where it acts as a specific uncoupler of photophosphorylation. Streptomycin inhibits chlorophyll synthesis and enhances anthocyanin accumulation. These results suggest that there is very little, if any, interaction between photosynthesis and ‘high-irradiance-reaction’ anthocyanin synthesis in cabbage and mustard seedlings.     

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.     

CONTROL BY PHYTOCHROME OF EXTENSION GROWTH and POLAROTROPISM IN CHLORONEMATA OF Funaria hygrometrica

Abstract— The photocontrol of extension growth and polarotropism has been investigated in chloronemata of the moss Funaria hygrometrica Hedw. When grown on a 12/12 h light/dark cycle, chloronemata show light-stimulated diurnal variations in elongation rate with no evidence of a circadian rhythm. Regulation of elongation by a low energy, photoreversible phytochrome mechanism was demonstrated by brief red (R) and far red (FR) irradiations given either at the end of the day (EOD) or 6 h later as a night break (NB). Night break irradiation with polarised R caused polarotropic reorientations of chloronemal growth also through a FR-reversible, low energy phytochrome mechanism. Such transformations of P_r to P_fr were accompanied by a 90° shift in the orientation of the phytochrome chromophore. Microbeam irradiation indicated that the phytochrome chromophores involved in both responses were predominantly, if not entirely, located in the tip of the apical chloronemal cell.     

A STUDY OF CHLOROPHYLL FLUORESCENCE IN VIVO BY A FLOW METHOD

Abstract— A flow method has been devised for the study of fluorescence transients during the induction period of photosynthetic systems. The principle of transformation of time-transients into space-transients is quantitatively worked out and the characteristics and mode of operation of the apparatus is described and illustrated with experimental results.
With this method spectroscopic and kinetic investigation of in vivo chlorophyll fluorescence have been undertaken. The fluorescence emission can be split into two componmts—a variable part and a constant part—with different spectral distributions. The excitation spectrum of the light induced fluorescence changes shows that the variable part is sensitized by system 11, whereas the constant part mainly sensitized by system II has a small component originating from system I. The light-induced fluorescence change decays in darkness with half-time of about 10 ^-2 s; this decay is highly sensitive to the action of inhibitors of oxygen evolution.
The flow method can be extended to measurements of oxygen, luminescence, absorption differences. It permits using high-intensity analytic beams with no actinic action; it is well suited for the integration of low level signals.     

INDUCTION OF LEAF UNROLLING BY PHYTOCHROME and ACETYLCHOLINE IN ETIOLATED WHEAT SEEDLINGS

Abstract-Phytochrome regulates the unrolling of primary leaf sections from 8-day-old dark-grown wheat ( Triticum aeslivum L. cv. Arminda) seedlings. Red light (R)-stimulated unrolling of leaf sections pretreated in 1 m M ethylene-bis-(β-aminoethylether)- N,N,N',N' -tetraacetic acid (EGTA) if 1 m M CaCl₂ was added during a 30 min treatment period including and following irradiation. Nifedipine at 1 μ M (a Ca²+-channel antagonist) applied 10 min before R prevented the R stimulation of leaf unrolling. The Ca²+-channel agonist Bay K-8644 (1 μ M ) and acetylcholine (ACh, 1 mY M ) stimulated unrolling of leaf sections prewashed in EGTA in darkness, if 1 m M CaCl₂ was present in the medium during a 30 min treatment period. Acetylcholine also induced leaf unrolling in the absence of Ca²+ when 100 μ M NaCl was present in the medium. Apart from ACh, only carbamylcholine out of the choline derivatives tested was active in induction of leaf unrolling in the presence of 1 m M Ca²+. The ACh receptor antagonists, atropine (10 μ M ) AND D-tubocurarine (10 μ M ), nullified the ACh-induced Ca²+- and Na+-dependent leaf unrolling, respectively. Muscarine and nicotine, agonists of ACh, at 1 μ M stimulated leaf unrolling in the presence of Ca²+ and Na+, respectively. The ACh-induced Ca²+-dependent leaf unrolling was reduced by 1 μ M Nifedipine, 10 μ M Li+ and 10 μ M "calmodulin" inhibitor, trifluoperazine (TFP), whereas only TFP was active in the reduction of the Na+-dependent ACh-induced leaf unrolling response. It is proposed that leaf unrolling of dark-grown primary wheat leaves can be regulated by phytochrome and by activation of two different types of ACh receptors.     

EFFECT OF CONTINUOUS FAR-RED PREIRRADIATION ON THE LAG PHASE OF CHLOROPHYLL A ACCUMULATION IN PHARBITIS NIL COTYLEDONS

In Pharbitis nil cotyledons, the lag phase of chlorophyll a (Chi a) accumulation which continues for about 2 h after the onset of continuous white light is eliminated by preirradiation with far-red light (FR) for 24 or 48 h. When the period of FR preirradiation is prolonged to 72 h or more, however, the lag phase is observed again (FR-induced lag phase) and the rate of Chi a accumulation during the rapid accumulation phase is lowered below the dark control level.
The application of exogenous 5-aminolevulinate (J-ALA) completely eliminates the FR-induced lag phase, but this treatment eliminates the normal lag phase in dark-grown cotyledons only partially (i.e. Chi a is accumulated but only slowly during the first few h). Application of a 5-ALA precursor, such as glycin + succinate. 2-ketoglutarate or glutamate, eliminates neither the FR-induced lag phase nor the normal lag phase.
A 24- or 48-h FR irradiation seems not only to enable the synthesis of 5-ALA but also to make the other regulatory factors favourable for Chi accumulation. When the period of FR irradiation is prolonged to 72 h or more, the ability to synthesize 5-ALA may be lost.     

PHOTOCONVERSION OF PHYTOCHROME IN VIVO STUDIED BY DOUBLE FLASH IRRADIATION IN Mougeotia AND Avena

Abstract— The kinetics of phytochrome phototransformation from the red-absorbing form (P_r) to the far-red-absorbing form (P_fr) in vivo at 22°C were studied using a double flash apparatus with 1-ms flashes. Photoconversion by simultaneous flashes of red light saturates at a low P_fr level, indicating the possible attainment of a photoequilibrium between the excitation of P_r and the photoreversion of intermediates in the course of the I-ms flashes. At saturation energy, simultaneous flashes resulted in about 50% as much P_fr as was produced by saturating irradiation with 5 s red light. Intermediates of the phototransformation pathway were analysed by separating two red or a red and a far-red flash by variable dark intervals. In both plants phototransformation intermediates with half-lives < 1 ms occur, but they are too short-lived to characterize by our method. The subsequent intermediates have half-lives of about 7 ms and 150 ms in A vena , 2 ms and 10 ms in Mougeotia. The conversion from P_r to P_fr seems to be completed 1 s after the red flash in Avena. In the alga Mougeotia , P_fr formation seems to be finished within only 50 ms after the inducing red flash. The kinetics obtained from physiological and spectrophotometric experiments with Avena mesocotyls are almost identical. These observations indicate that the physiological response corresponds directly to the amount of P_fr produced and not to phototransformation intermediates or "cycling" between P_r and P_fr.     

QUENCHING OF CHLOROPHYLL FLUORESCENCE BY NITROBENZENE

Abstract—Nitrobenzene quenching of chlorophyll fluorescence in ethanol has been investigated. Steady state relative quantum yields have been measured and fluorescence decay rates were determined using both nanosecond photon counting and picosecond pulses from a mode-locked Nd³⁺ glass laser.
The fluorescence decay is described by
1( t )= I ₀ exp (- t/τ−At^1/2 )
the form predicted for decay governed by the kinetics of the continuum model of diffusion controlled reactions. From the parameters of the fluorescence decay, the encounter distance is 5–7 A° the mutual diffusion coefficient is 0.62 × 10^--⁵ cm2s^-1± 12%.
Some of the fluorescence quenching is also attributed to static quenching by a nitrobenzene-chlorophyll, ground-state complex. The equilibrium constant for formation of this ground-state complex was determined to be 4.1 M ^-1. The combined dynamic and static quenching model allows calculation of quantum yields of fluorescence in good agreement with the experimentally determined quantum yields.     

PHYTOCHROME INTERMEDIATES IN VIVO–II. CHARACTERISATION OF INTERMEDIATES BY DIFFERENCE SPECTROPHOTOMETRY

Abstract— In epicotyl tissue of Pisum , irradiation of P_r at – 196°C forms a stable product P₆₉₈, whereas P_fr forms a stable product P₆₅₀. On warming P₆₉₈, dark transformation to P_r predominates. On warming P₆₅₀ to – 70°C an intermediate P₆₉₀ is formed which bleaches on further warming to –10°C. When tissue is cooled to –196°C during actinic irradiation, difference spectra for subsequent warming to –10°C indicate that P_r, P_fr and an intermediate P₇₁₀ are formed from weakly absorbing intermediates. Complete photoconversion of P_r to P_fr is not possible at temperatures below –5°C. As the temperature is reduced, the amount of P_fr produced from P_r decreases, while P₇₁₀ increases. P₇₁₀ can be photoconverted at –20°C and above, ultimately forming P_r, but in contrast to P_fr it is not photoconvertible at –196°C.     

LASER PHOTOLYSIS STUDIES OF QUINONE REDUCTION BY CHLOROPHYLL a IN ALCOHOL SOLUTION

Upon laser photolysis of chlorophyll-quinone solutions in ethanol, transients due to the chlorophyll triplet state (C_t), the chlorophyll cation radical (C⁺) and the semiquinone radical (Q^-) can be observed. The rise of Q^- parallels the decay of C_t. demonstrating the precursor role of the triplet. The decay of C⁺ is second order, consistent with reverse electron transfer, and has a rate constant which is independent of quinone potential, and an activation energy of 14kJ/mol due mainly to the temperature dependence of solvent viscosity. Triplet quenching and C⁺ yield are found to decrease with decreasing quinone potential.     

原子吸收光谱法间接测定绿茶叶中叶绿素总量

以0.4 mol.L-1盐酸7.5 mL为提取剂,经过两次各15 s中档超声波辅助酸置换出卟啉环中的镁离子,原子吸收光谱法间接测定了不同采摘期的绿茶叶片中叶绿素总含量。方法的加标回收率为98%~99%,RSD为2.3%~4.2%。     

PHYTOCHROME INTERMEDIATES IN VIVO-I. EFFECTS OF TEMPERATURE, LIGHT INTENSITY, WAVELENGTH AND OXYGEN ON INTERMEDIATE ACCUMULATION

Abstract— Accumulation of weakly absorbing phytochrome intermediates has been demonstrated in Pisum epicotyl tissue under conditions of pigment cycling using a quasi-continuous measuring spectrophotometer. An action spectrum shows 690–700 nm to be the most efficient wavelength range in this process. Difference spectra for the decay of intermediates maintained by 690 nm light show that, if the experiment is done at 0°C, only P_fr is formed. At – 11°C, intermediates decaying to P_r can also be observed. At – 20°C, P_r is produced as well as a pigment with peak absorption at 710nm. Kinetic analysis of intermediate decay at – 11°C reveals that at least two intermediates are maintained by 690 nm light. The level of intermediate maintained by incandescent light at 0°C was 25% higher in air than in nitrogen.