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.     

PHOTOREGULATION OF NITRATE UTILIZATION IN GREEN ALGAE AND HIGHER PLANTS*

Abstract— Nitrate reductase from eukaryotes can be reversibly inactivated, blue light being an effective activating agent both in vitro and in vivo. Hydroxylamine proved to be a powerful inactivating agent of Ankistrodesmus braunii nitrate reductase. Irradiation with blue light of NH₂OH-inactivated nitrate reductase, specially in the presence of μM amounts of FAD, promoted the recovery of the enzyme activity. Similarly, photoexcited methylene blue reactivated spinach nitrate reductase. On the other hand, in vitro nitrate reductase is highly susceptible to photodynamic inactivation caused by singlet O₂. Aerobic incubation of the active spinach enzyme with either FMN or methylene blue under either blue or red light respectively led to its irreversible inactivation. Irradiation of frozen and thawed spinach leaf discs also promoted, in situ, an irreversible inactivation of nitrate reductase, provided that 6₂ was present in the incubation mixture. Thus, either in vitro or in situ, light can cause two quite different responses of nitrate reductase, its blue light-dependent photoactivation in a flavin sensitized reaction and its photodynamic inactivation in a singlet O₂-dependent process.     

INTERACTION BETWEEN CHLOROPLAST BIOGENESIS and PHOTOREGULATION OF AMYLASES IN Pennisetum americanum LEAVES*

In pearl millet (Pennisetum americanum) seedlings, light acting via phytochrome stimulates amylase activity in the leaves. The maximum photostimulation of amylase is observed in the segment proximal to the leaf base. This photostimulation mainly results from an enhancement in β-amylase activity (EC 3.2.1.2.), which constitutes80–90% of leaf amylase activity. The subcellular fractionation studies revealed that while p-amylase is localized only in the extraplastidic compartment, a-amylase (EC 3.2.1.1.) is localized both in the cytosol and in the chloroplasts. The mesophyll cells contained both a- and β-amylase activity, but only a-amylase activity was present in the bundle sheath cells. The Norflurazon-mediated photooxidation of chloroplasts during leaf development also eliminated plastidic α-amylase activity, confirming dependence of its photoregulation on the chloroplast biogenesis.     

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.     

SPECIFIC PHOTOREGULATION BY PHYTOCHROME OF EPINASTY and LIGHT-INDUCED ETHYLENE PRODUCTION IN MARCHANTIA POLYMORPHA*

Abstract— A study is made of the influence of phytochrome on light-induced ethylene production and epinasty in green Marchantia polymorpha thalli. Ethylene production of thalli irradiated with terminal far-red is not substantially affected by the presence or absence of CO₂, in contrast with the controls which show a clearcut CO₂ dependency. Photoreversibility and fiuence response data indicate the involvement of the low energy red'far-red reversible type of phytochrome action for both the light-induced ethylene production and the control of epinasty. The far-red effect is further characterized by a difference in escape from reversibility between light-induced ethylene production and the elimination of epinasty.     

PHOTOCONTROL OF ANTHOCYANIN SYNTHESIS IN TOMATO SEEDLINGS: A GENETIC APPROACH*

The photocontrol of anthocyanin synthesis in dark-grown seedlings of tomato (Lycopersicon esculentum Mill.) has been studied in an aurea (au) mutant which is deficient in the labile type of phytochrome, a high pigment (hp) mutant which has the wild-type level of phytochrome and the double mutant au/hp , as well as the wild type. The hp mutant demonstrates phytochrome control of anthocyanin synthesis in response to a single red light (RL) pulse, whereas there is no measurable response in the wild type and au mutant. After pretreatment with 12 h blue light (BL) the phytochrome regulation of anthocyanin synthesis is 10-fold higher in the hp mutant than in the wild type, whilst no anthocyanin is detectable in the au mutant, thus suggesting that it is the labile pool of phytochrome which regulates anthocyanin synthesis. The au/hp double mutant exhibits a small (3% of that in the hp mutant) RL/far-red light (FR)-reversible regulation of anthocyanin synthesis following a BL pretreatment. It is proposed that the hp mutant is hypersensitive to the FR-absorbing form of phytochrome (P_fr) and that this (hypersensitivity) establishes response to the low level of P_fl. (below detection limits in phytochrome assays) in the au/hp double mutant.     

PHYTOCHROME INDUCTION OF PHOTOREACTIVATING ENZYME IN Phaseolus vulgaris L. SEEDLINGS*

Abstract— Photoreactivating enzyme (PRE) activity was measured in hypocotyls of Phaseolus vulgaris L. seedlings using a radioimmunoassay for thymine dimers. In dark-grown seedlings a five-fold increase in PRE activity was observed after 6 h of irradiation with blue or far-red light. Short time irradiations with red light were also effective. Reversibility of this red-light-effect by a subsequent short term irradiation with far-red light and also the high effectiveness of continuous far-red light indicate that PRE activity is under phytochrome control. This observation points to PRE induction via gene activation.     

PHOTOREGULATION OF α-CHYMOTRYPSIN ACTIVITY IN ORGANIC MEDIA: EFFECTS OF BIOIMPRINTING

Abstract α-Chymotrypsin exhibits photoswitchable activities in an organic solvent after covalent modification of the protein backbone with thiophenefulgide active ester (2). The thiophenefulgide-modified α-chymotrypsin exhibits reversible photoisomerizable properties between states (3)-E and (3)-C. The modified α-chymotrypsin, where nine lysine residues are substituted by thiophenefulgide units, retains 60% of the activity of the native enzyme. The activities of thiophenefulgide-modified α-chymotrypsin toward esterification of N -acetyl-L-phenylalanine (4) by ethanol in cyclohexane are controlled by the configuration of the attached photoisomerizable component and by prior bioimprinting of the protein backbone with the reaction substrate (4). The esterification of (4) in cyclohexane using bioimprinted (3)-C is two-fold faster than in the presence of (3)-E. In the presence of a nonbioimprinted enzyme, esterification of (4) by (3)-C is five-fold faster than with (3)-E. The activity of bioimprinted (3)-E toward esterification of (4) is 4.5-fold higher than that of nonbioimprinted (3)-E. Switchable cyclic esterification of (4) is accomplished by sequential photoisomerization of the thiophenefulgide-modified α-chymotrypsin between states (3)-C and (3)-E.     

ON THE POLARITY OF PHYTOCHROME CONTROLLED LEAF UNFOLDING IN DARK-GROWN SEEDLINGS OF TRITICUM AESTIVUM*

Abstract— The apical ends of sections cut from etiolated primary wheat leaves show a higher degree of phytochrome-mediated leaf unfolding than the basal ends, regardless of the position of the segment in the leaf. It could be clarified that this polarity is not the result of a stable gradient within the intact dark grown leaf. Partial irradiation experiments have shown that leaf unfolding can be induced much more effectively by an irradiation of the apical half of a section than of the basal half. Though only the basal end of the sections was incubated with gibberellic acid (GA₃) in darkness, the degree of the GA₃-induced leaf unfolding was highest at the apical end of these non-irradiated segments. The results suggest that in each separate leaf section a new gradient of a still unknown “unfolding-factor” is established.     

THE EFFECT OF PROLONGED LIGHT EXPOSURE ON THE EFFECTIVENESS OF PHYTOCHROME IN ANTHOCYANIN SYNTHESIS IN TOMATO SEEDLINGS*

Abstract— The hypocotyl of the tomato ( Lycopersicon esculentum ) seedling synthesizes large amounts of anthocyanin if exposed to prolonged light. Single light pulses are totally ineffective. The involvement of phytochrome can be shown by light pulse treatments following a prolonged light exposure. It is predominantly the action of blue/UV light which leads to a high responsiveness of anthocyanin synthesis towards phytochrome. Moreover, the data suggest a phytochrome-independent action of blue/UV light, in particular of UV-B, on anthocyanin synthesis.     

ON THE POLARITY OF GREENING IN PRIMARY LEAVES OF DARK AND LIGHT-GROWN SEEDLINGS OF Triticum aestivum*

Abstract— In primary leaves of four-day-old dark-grown wheat seedlings the capability to accumulate chlorophyll under white light is highest in the tissues of the apical region. It declines though, as the dark-grown seedling ages and the primary leaf grows longer. This decrease is lower in the basal than in the apical regions of a leaf, which results in an inverted polarity of the capability to accumulate chlorophyll If the seedlings are grown under continuous white light applied from four different directions, the high capability of greening observed in the apical tissues of young leaves stays at that level even in old seedlings: the polarity does not change during culturing. The saturation-level of greening is much higher in the tissues taken from the apical region of a primary leaf than in those from the basal region.     

TWO SPECTROSCOPICALLY AND PHOTOCHEMICALLY DISTINGUISHABLE PHYTOCHROMES IN ETIOLATED SEEDLINGS OF MONOCOTS AND DICOTS *

Abstract Fluorescence (F) emission spectra of the red-absorbing phytochrome form (Pr) at 85 K, temperature dependence of the F intensity and the extent of the Pr F changes in the phototransformation of Pr into the first stable photoproduct (lumi-R) at 85 K (γ₁,) and into the far-red-absorbing form (Pfr) at 267 K (γ₂) were investigated in etiolated shoots and roots of monocots (oat, maize, rice) and dicots (pea, cress). These characteristics monotonously changed as a function of the phytochrome content, [Ptot]: with its decrease to 3-5% of the maximal values, the F spectrum shifts from 686 nm to 682 nm, its half-band width rises from 22 nm to ca 25 nm, temperature dependence of Pr F changes its character, γ¹, drops down from ca > 0.45 to ca 0.05-0.10 and γ₂ from 0.80–0.82 to ≤0.70. These data were interpreted in terms of two different phytochromes whose relative concentration varies with [Ptot]: (1) a longer wavelength type with the F maximum at 686 nm, low activation energy of the photoreaction (Ea ≤ 3–4 kj/ mol) and high extent of the phototransformation at 85 K (0.49 ± 0.03) and at 267 K (ca 0.85) (Pra); (2) a shorter wavelength type practically inactive at 85 K with F maximum at 682 nm, higher Ea (ca 35 kj/mol) and lower extent of the Pr & Pfr phototransformation (≤0.70) (Pri). [Pra] widely varies in different parts of the seedlings (up to 100 times) and Pra dominates when [Ptot] is high. The [Pri] is much more constant (variations, <10 times), and it becomes the major one when [Ptot] drops down. The two species are likely to belong to the labile (type 1) and stable pools of pigment and not to be connected with the localization of the pigment in the cell since red-far-red preillu-mination, which is believed to bring about sequestering of the pigment, does not change their relative concentration and properties.     

PHOTOREGULATION OF α-CHYMOTRYPSIN ACTIVITY BY ISOMERIZABLE INHIBITORS. STUDY OF THE ACTION MECHANISM*

Abstract— The N- p -phenylazophenyl-N-phenylcarbamyl chloride (PAPC) in its cis form is five times more active as inhibitor of α-chymotrypsin than in the trans form. In the present work, derivatives of PAPC have been synthesized. Each of these new compounds is photoisomerizable and is an inhibitor (in the cis and in the trans form) of α-chymotrypsin. The cis isomer is always more active than the trans. The m -methyl derivative is 17.5 times more active in the cis form than in the trans , whereas, for the para -substituted compound, this ratio is only 3.5.
Several hypotheses can explain this difference of activity between the cis and trans isomers: (1) steric hindrance towards the trans isomer, (2) lower affinity of the enzyme for the trans isomer, (3) higher reactivity of the complex formed between the enzyme and the cis form of the inhibitor. These hypotheses are discussed.     

IN VITRO SYNTHESIS OF PHYTOCHROME APOPROTEIN DIRECTED BY mRNA FROM LIGHT AND DARK GROWN AVENA SEEDLINGS*

Abstract— The aim of this work was to compare the translatability of poly-A-RNA from light- and dark-grown Arena seedlings, the product of which should be phytochrome. Polysomal poly-A-RNA was isolated from 4-day-old dark-grown Avena seedlings (Avena sativa L. cv. Garry) and translated in a rabbit reticulocyte lysate system. Immunoprecipitation by anti-phytochrome serum was used for measurements of specific phytochrome translation. Characterization of the translated and immunoprectpitated protein was performed by comparison with [₃₅S]-methionine in vivo-labelled phytochrome. The specificity of the precipitation was shown by parallel use of non-specific serum and by competitive inhibition of precipitation by exogenous unlabelled phytochrome isolated by affinity chromatography.     

火焰原子吸收光谱法测定丹参及复方丹参注射液中微量元素

用硝酸高氯酸混酸(2．4：1)对丹参及复方丹参注射液进行消化处理，用火焰原子吸收光谱法测定处理溶液中的铜、镉、钙、铁、镁、镍、铅、锌、钴、锰等10种微量元素。回收率为97．0％～103．0％，相对标准偏差为1．8％～4．6％。结果表明，丹参及复方丹参注射液中对人体有益的元素如铁，镁，锌含量丰富，具有很大的使用价值。     

CHANGES IN THE RATES OF PHOTOCONVERSION OF PHYTOCHROME DURING ETIOLATION IN MUSTARD SEEDLINGS

Abstract— The relative phytochrome photoconversion rates in cotyledons and hypocotylar hook of etiolating mustard ( Sinapis alba L.) seedlings were measured between 16 and 96 h after sowing. It was found that at constant fluence rates photoconversion rate in red light increases in both organs with time whereas the photoconversion rate in far-red (756 nm) light decreases with time of development. Since the isosbestic point remains constant, it was concluded that the observed changes cannot be attributed to changes of extinction coefficients. It was not possible, however, to decide whether the observed changes are due to changes of light attenuation or quantum yields.     

RELATIVE IMPORTANCE OF BLUE LIGHT AND LIGHT ABSORBED BY PHYTOCHROME IN GROWTH OF MUSTARD (Sinapis alba L.) SEEDLINGS*

Abstract— Hypocotyl straight growth in mustard (Sinapis alba L.) responds very strongly and in precisely the same way to low fluence rate red (RL) and white light (WL). The effect of weak light can be attributed fully to light absorption by phytochrome. Only with increasing fluence rate an effect of blue light (BL) comes into play which cannot be explained by the action of phytochrome. However, this specific action of BL can be demonstrated in hypocotyl growth of mustard seedlings only up to 5 days after sowing (25°C). With older seedlings control of hypocotyl growth seems to be exerted exclusively via phytochrome. Regarding the far-red light dependent “high irradiance reaction” (FR-HIR) it was found that it plays a dominant role in growth of mustard only during a relatively short period. It tends to disappear in favor of a RL-HIR between 3 and 4 days after sowing. It is concluded that the seedling exhibits a largely endogenous temporal pattern of responsiveness to light. Phototropism of the mustard seedling can be elicited by low fluence rates (< 1 mW m^?2) of unilateral BL. This same light has no effect on straight growth. It is concluded that BL-dependent phototropic growth response of a hypocotyl and the effect of BL on longitudinal growth of the hypocotyl are unrelated phenomena.     

PHOTOBIOLOGICAL CHARACTERIZATION OF A SPORE GERMINATION MUTANT dkgl WITH REVERSED PHOTOREGULATION IN THE FERN Ceratopteris richardii

Abstract— This paper describes the mutant dkgl in the fern Ceratopteris richardii, which shows rapid germination in darkness but is markedly inhibited by white light. Action spectra plotted at 10 nm intervals from 400 to 800 nm are presented for germination responses of wild-type and mutant spores to photon flux densities of 0.004, 0.04 and 0.4 jtmol/mVs. The action spectra for wild-type spores exhibit a sharp phytochrome-mediated peak at 660 nm, a broad peak from 670 to 740 nm resulting from an apparent high irradiance response and no germination below 560 nm. In the corresponding action spectra for mutant spores, the blue region displays rather complex fine structure with prominent minima at 450 and 470 nm, which suggests that cryptochrome is unaltered in these spores. The region from 550 to 640 nm shows the greatest inhibition of spore germination, but this region exhibits no obvious fine structure, which argues rather strongly against the possibility of a unique photoreceptor being active in mutant spores. The mutant spectra resemble the wild-type spectra in the region from 650 to 800 nm, and thus phytochrome seems normal in the mutant spores. The dkgl mutation appears to act late in the phytochrome transduction pathway where a hypothetical coupling protein may regulate the light-sensitive step in spore germination.     

CIRCANNUAL COURSE OF PHOTOMORPHOGENETIC REACTIVITY IN ETIOLATED BEAN SEEDLINGS

Hook opening and leaf expansion, measured 24 h after standard red light illumination, were considered as parameters determining the photoactivity of etiolated Phaseolus vulgaris L. seedlings. The experiment was repeated every h during one 17-h period each month for 1 year. The mean values for each experimental period indicated that the photoactivity of the etiolated seedlings changed markedly throughout the year according to a seasonal pattern.