Action spectra for chromatic adaptation in the blue-green alga Fremyella diplosiphon

Action spectra for chromatic adaptation in Fremyella diplosiphon Drouet have been determined using techniques previously described. Action maxima are at 540 nm, with a half-band width of 80 nm, for induction of phycoerythrin synthesis (green action) and at 650 nm, with a half-band width of 90 nm, for reversal of induction of phycoerythrin synthesis (red action). The red-action spectrum includes a secondary action band centered at ca. 360 nm. Red and green action overlap from 570 to 590 nm with an isosbestic point in the vicinity of 580 nm. Shoulders are present at 520 and 630 nm. Red light is more active than green light. The 540:650-nm quantum effectiveness ratio is 1:7. There is relatively little action of either kind in the blue. The 387:540 nm and 460:650-nm quantum effectiveness ratios are zero. These results contrast strongly with previous determinations in the same organism, with major activity indicated in the blue; they are consistent with the control of photomorphogenesis in the Cyanophyta by a master pigment, analogous to phytochrome.Abbreviations APC allophycocyanin - PC physocyanin - PE phycoerythrin     

Rat carotid body chemosensory discharge and glomus cell HIF-1 alpha expression in vitro: regulation by a common oxygen sensor

Addition of Pco ( approximately 350 Torr) to a normoxic medium (Po(2) of approximately 130 Torr) was used to investigate the relationship between carotid body (CB) sensory discharge and expression of hypoxia-inducible factor 1 alpha (HIF-1 alpha) in glomus cells. Afferent electrical activity measured for in vitro-perfused rat CB increased rapidly (1-2 s) with addition of high CO (Pco of approximately 350 Torr; Po(2) of approximately 130 Torr), and this increase was fully reversed by white light. At submaximal light intensities, the extent of reversal was much greater for monochromatic light at 430 and 590 nm than for light at 450, 550, and 610 nm. This wavelength dependence is consistent with the action spectrum of the CO compound of mitochondrial cytochrome a(3). Interestingly, when isolated glomus cells cultured for 45 min in the presence of high CO (Pco of approximately 350 Torr; Po(2) of approximately 130 Torr) in the dark, the levels of HIF-1 alpha, which turn over slowly (many minutes), increased. This increase was not observed if the cells were illuminated with white light during the incubation. Monochromatic light at 430- and 590-nm light was much more effective than that at 450, 550, and 610 nm in blocking the CO-induced increase in HIF-1 alpha, as was the case for chemoreceptor discharge. Although the changes in HIF-1 alpha take minutes and those for CB neural activity occur in 1-2 s, the similar responses to CO and light suggest that the oxygen sensor is the same (mitochondrial cytochrome a(3)).     

The Effect of Light on the Phytolization of Chlorophyllide α and the Spectral Dependence of the Process

Seven days old dark-grown etiolated seedlings of wheat cultivated on nutrition solution were given short light impulses of high intensity followed by a dark period, during which the phytolization of chlorophyllic α was studied. If the leaves prior to the short light impulses were given a pretreatment consisting of a light impulse followed by three hours of darkness the phytolization rate increased. Monochromatic light as pretreatment gave a response which was mainly restricted to the red region with a peak around 660 nm.     

Influence of spectral wavelength and N:P ratio on the cell cycle of Synechococcus strain CSIRNIO1 isolated from the central Eastern Arabian Sea

The novel phycoerythrin-containing Synechococcus strain CSIRNIO1 belonging to phylogenetic clade II was isolated from the coastal Arabian Sea. Chromophore characteristics of this isolate revealed the presence of phycoerythrin I (PEI), which allows it to utilize green light efficiently. The DNA distribution data indicate a bimodal slow growth model synchronized with the light/dark cycle. The duration of the cell cycle was regulated by spectral wavelength and nutrient concentration. Nitrate and phosphate enrichment shortened G1 phase duration when cells were exposed to equal doses of photosynthetically usable radiation (PUR) of different spectral wavelengths. G2 phase duration was influenced by spectral quality and phosphate concentration. S phase duration was not affected by the spectral wavelength. However, a shorter doubling time corresponding to shortened G1 and S phases was observed under nitrate enrichment. Phosphate enrichment resulted in shortening of all three phases (G1, S and G2). More efficient utilization of green and red light than blue light regulated the duration of the cell cycle as well as the doubling time, suggesting spectral selectivity in this strain. The effects of spectral wavelengths under varying nutrient concentrations will determine the proliferation of Synechococcus and its adaptation to different environmental conditions.     

Effect of infrared and visible light on 2-azidoanthraquinone in the QA binding site of photosynthetic reaction centres. An unusual mode of activation of a photoaffinity label

Quinone-depleted reaction centres of Rhodobacter sphaeroides were reconstituted with 2-azidoanthraquinone and irradiated with short (50 ms) pulses of intense infrared (lambda = 850 +/- 50 nm) or visible light (460 less than lambda less than 630 nm). The irradiations brought about the rapid degradation of the protein-bound photoaffinity label even though it does not absorb light in either spectral region. The decomposition of the label was accompanied by a covalent modification of subunit M and by a loss of photochemical activity of the reaction centre protein (as measured by the light-induced electron transfer onto the primary acceptor, QA). In the case of the photolysis with IR light, these effects were triggered by the reduction of the protein-bound quinone (QA) to the semiquinone (Q-A) in the process of primary charge separation. The resulting reactive species showed properties of both a semiquinone and a triplet nitrene. Efficiency and specificity of the covalent incorporation were markedly improved when visible rather than IR light was used for the photolysis, presumably, because the triplet nitrene resulting from the primary charge separation was further activated in a second light-dependent reaction. The results suggest that, in conventional photoaffinity labeling experiments, the efficiency and specificity of the covalent incorporation of an aryl azide photolabel into a target protein may be improved when the photolysis is carried out with a combination of UV and intense visible light, rather than with UV light alone.     

Phycocyanin synthesis and degradation in the blue-green bacterium Anacystis nidulans.

Cellular content and rates of synthesis of the apoprotein subunits of phycocyanin in Anacystis nidulans cultures undergoing, and recovering from, nitrate starvation were measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total and immunoprecipitable soluble proteins. Results indicated that (i) nitrate starvation provokes coordinate degradation of apoprotein subunits: (ii) de novo synthesis of these subunits is selectively depressed during starvation; (iii) nitrate restoration provokes coordinate increases in the rates of synthesis of these subunits, although maximal rates are not achieved for 6 to 10 h after readdition of nitrate; and (iv) illumination affects both relative and absolute rates of apoprotein formation.     

Exciton interactions in phycoerythrin

Upon assembly of the phycoerythrin trimer into hexamer and the hexamer into dodecamer, marked spectral changes are observed. The absorption and circular dichroism spectra of the various phycoerythrin aggregates were resolved into Gaussian components representing individual electronic transitions of phycoerythrobilin chromophores within these proteins. While the contribution of a broad, sensitizing band (at 525 nm) is constant, with increasing aggregate size, a short-wavelength pair of bands centered at 555 nm decreases concomitantly with a dramatic increase in the intensity of a long-wavelength pair of chromophore transitions centered at 563 nm. The implications of these spectral changes for efficient energy transfer in the phycobilisome are discussed.     

Light Rquirements for Photoperiodic Sensitivity in Cotyledons of Dark-grown Pharbitis nil

The light requirements for induction of flowering by a long dark period were investigated in dark-grown seedlings of Pharbitis nil Chois, cv. Violet. The cotyledons bcame photoperiodically sensitive to a 24 h dark period by two 1 min red irradiations (6.3 μmol m⁻² S⁻¹) separated by a 24 h dark period. The reversibility of the effect of brief red irradiations, and the effectiveness of low energies of red irradiation suggest the involvement of phytochrome in the induction of photoperiodic sensitivity. Partial de-etiolation occurred after these brief periods of red irradiation but the seedlings were not capable of net CO₂ uptakeeven 7 h after the start of the main light period that followed the critical dark period. A changing response to the duration of the priod of darkness given between the two short red irradiations showed the the correct phasing of an endogenous photoperiodic rhythm is needed for the attainment of photoperiodic snsitivity.     

Effect of bacteriophage lambda infection on synthesis of groE protein and other Escherichia coli proteins.

We used two-dimensional gel electrophoresis to quantitate the changes in rates of synthesis that follow phage lambda infection for 21 Escherichia coli proteins, including groE and dnaK proteins. Although total protein synthesis and the rates of synthesis of most individual E. coli proteins decreased after infection, some proteins, including groE protein, dnaK protein, and stringent starvation protein, showed increases to rates substantially above their preinfection rates. Infection by lambda Q- affected host synthesis in the same way as infection by gamma+, whereas infection by lambda N- showed no detectable effect on host synthesis. Deletion of the early genes between att and N abolished the effect, and shorter deletions in this region gave intermediate effects. By this sort of deletion mapping, we show that a large part, though not all, of the effect of lambda infection on host protein synthesis can be ascribed to the early region that contains phage genes Ea10 and ral. We compared the changes in protein synthesis after infection with the changes that occur in uninfected cells upon heat shock or amino acid starvation. The spectrum of changes that occurred on infection was very different from that seen after heat shock but quite similar to that seen during amino acid starvation. Despite this similarity of the effects of lambda infection and starvation, we did not detect any increase in the level of guanosine tetraphosphate during infection. We show that the groE protein is the same protein as B56.5 of Lemaux et al. (Cell 13:427-434, 1978) and A protein of Subramanian et al. (Eur. J. Biochem. 67:591-601, 1976).     

The characteristics of light in floral induction in vitro of Cichorium intybus. The possible role of phytochrome

The action of light in the initiation of floral buds in vitro has been studied using monochromatic light qualities on root explants of a long day plant, Cichorium intybus L. cv. Witloof. Red light (660 nm, 0.30 W m^-2) promotes flowering, while far-red (730 nm, 0.31 W m^-2) and irradiation with combined red + far-red (0.20 + 0.41 W m^-2) have no effect. In short day conditions floral response can be obtained in two ways: 1) by interrupting the dark period with 5 brief irradiations of red light (0.45 W m^-2, 12 min) at regular intervals, although these are counteracted by far-red irradiations of equal intensity and duration; 2) by interrupting the long night with 5 h red light applied during the second third of the night, while at the beginning or at the end it is ineffective. Red light efficiency appears to depend on the photosynthetic activity of the tissues, so that flowering increases with increasing intensity of white light and is suppressed if no white light is supplied. The reproductive development is determined by the coordination of proper irradiation conditions with sufficient sensitivity of the perceiving meristematic cells. The period of highest sensitivity to environmental light conditions in the life cycle of a Cichorium root explant occurs between the 8th and the 16th day after the start of the culture. The data strongly suggest that phytochrome is involved in flower induction of Cichorium in vitro.     

Picosecond time-resolved fluorescence spectroscopy of K-590 in the bacteriorhodopsin photocycle.

The fluorescence spectrum of a distinct isometric and conformational intermediate formed on the 10(-11) s time scale during the bacteriorhodopsin (BR) photocycle is observed at room temperature using a two laser, pump-probe technique with picosecond time resolution. The BR photocycle is initiated by pulsed (8 ps) excitation at 565 nm, whereas the fluorescence is generated by 4-ps laser pulses at 590 nm. The unstructured fluorescence extends from 650 to 880 nm and appears in the same general spectral region as the fluorescence spectrum assigned to BR-570. The transient fluorescence spectrum can be distinguished from that assigned to BR-570 by a larger emission quantum yield (approximately twice that of BR-570) and by a maximum intensity near 731 nm (shifted 17 nm to higher energy from the maximum of the BR-570 fluorescence spectrum). The fluorescence spectrum of BR-570 only is measured with low energy, picosecond pulsed excitation at 590 nm and is in good agreement with recent data in the literature. The assignment of the transient fluorescence spectrum to the K-590 intermediate is based on its appearance at time delays longer than 40 ps. The K-590 fluorescence spectrum remains unchanged over the entire 40-100-ps interval. The relevance of these fluorescence data with respect to the molecular mechanism used to model the primary processes in the BR photocycle also is discussed.     

Spectral correspondence between visual spectral sensitivity and bioluminescence emission spectra in the click beetle Pyrophorus punctatissimus (Coleoptera: Elateridae)

The presence of two spectral mechanisms, near-ultraviolet and green (lambda(max)=545nm), is strongly suggested by electroretinographic visual spectral sensitivity curves obtained under dark and red chromatic adaptation conditions in the compound eyes of the click beetle Pyrophorus punctatissimus. The bioluminescence emission of the dorsal prothoracic lanterns is deep green (lambda(max)=543nm) and that of the ventral abdominal lantern is lime green (lambda(max)=556nm) in colour in P. punctatissimus. A broad green visual receptor would detect both deep green and lime green bioluminescent optical signals.     

Reaction of cyanide with cytochrome aa3 in isolated perfused rat head in situ.

The reaction of cyanide with cytochrome aa3 in intact mitochondria is known to differ significantly from the reaction with the isolated enzyme. To examine the cyanide reaction with cytochrome aa3 in situ, we studied the spectral characteristics and the reaction kinetics of cyanide with reduced brain cytochrome aa3 in an isolated perfused rat head preparation. Anaesthetized rats underwent bilateral carotid-arterial cannulation. The head (skull intact, muscle removed) was perfused with a crystalloid solution containing Na2S2O4, and the animal was then decapitated. By means of reflectance spectrophotometry the reaction of cyanide with cytochrome aa3 was continuously monitored with the use of the 590 nm-575 nm, 610 nm-575 nm and 590 nm-610 nm wavelength pairs. We found that: the kinetics of the absorbance change at 590 nm and 610 nm were similar, with almost identical apparent rate constants, suggesting that these spectral changes are the results of the formation of a single complex; the difference spectrum obtained on addition of cyanide to the fully reduced preparation showed a peak at 588 nm and a trough at 610 nm, consistent with spectral characteristics of the cyanide-ferrocytochrome aa3 complex in isolated enzyme and isolated mitochondria in vitro; this observation underscores the accuracy of monitoring the effects of inhibitors of mitochondrial function on cytochrome redox reactions in situ; the half-maximal (K0.5) effect was approx. 50 microM, significantly lower than that in vitro. The lower apparent K0.5 for cyanide in this preparation in situ may be due to a difference in the pH of the two systems. This approach provides the means to study the inhibitors of mitochondrial function in intact brain under a physiological environment.     

Inheritance of the replication complex by one of two daughter copies during lambda plasmid replication in Escherichia coli.

Direct measurement of DNA synthesis confirmed that lambda plasmid replication proceeds for several hours in an amino acid-starved relA mutant of Escherichia coli, leading to plasmid amplification; this replication is lambda cro-independent, but requires the function of lambda O initiator in the absence of its synthesis. This suggests that after the assembly of the replication complex (RC) at ori lambda the lambda O protein remains in this structure and the affinity of lambda O to ori lambda is alleviated in the assembled RC allowing its movement along the DNA. During amino acid starvation the lambda plasmid DNA synthesis per bacterial mass occurs at a constant level, as would be expected if the number of functioning RCs remained constant. This favors the idea that under these conditions the next replication round operates due to the activity of the RC inherited from the preceding round. Density shift experiments reveal indeed that, from two daughter plasmid copies synthesized after the onset of amino acid starvation only one is able to enter into the next round of replication. We infer that this is the plasmid copy that inherits the lambda O-enclosing RC from the previous replication round. Moreover, the same results of density shift experiments were obtained for plasmids synthesized before the onset of amino acid starvation. Therefore, we presume that in lambda plasmid-harboring bacteria growing in nutrient medium, every second plasmid circle bears an RC that originates from the preceding round of replication. This structure has to be assembled de novo only on the daughter plasmid copy that does not inherit the parental RC. In the absence of lambda O initiator synthesis in amino acid-starved relA cells this process cannot occur, leaving as the only replication pathway that driven by the parental RC. Our results are discussed in relation to the model of regulation of lambda plasmid replication.     

Spectral Dependence of Night-break Effect on Photoperiodic Floral Induction in Lemna paucicostata 441

A single dark period of longer than 8 hr induced flowering inLemna paucicostata 441 cultured in E medium. Monochromatic lightsof 450, 550, 650 and 750 nm with a half-power bandwidth of 9nm given for 10 min at the 8th hour of a 14-hr dark period inhibitedflowering. The fluence rates required for 50% inhibition were10, 0.5, 0.1 and 3 µmol m^–2. sec^–1, respectively.When applied between the 4th and the 10th hour of the dark period,lights of 450, 550 and 650 nm were inhibitory showing a maximumeffect at the 8th hour. But 750-nm light completely inhibitedflowering when applied at any time during the first 8 hr ofthe dark period. The inhibitory effect of 750-nm light givenat the beginning of the dark period was totally reversed bya subsequent exposure to 650-nm light, and the fluence-responsecurves for the effect of 750-nm light given at the 0, 4th and8th hour were essentially the same. This suggests that the presenceof P_FR is crucial for the floral initiation throughout the first8 hr of the inductive dark period. The role of phytochrome inthe photoperiodic flower induction of L. paucicostata is discussed. (Received January 4, 1982; Accepted March 19, 1982)     

Factors affecting the induction and release of secondary dormancy in Kalanchoë seeds

Abstract. Several short daily R irradiations are required from the first day of incubation on water to induce germination of Kalanchoë seeds. When the same light treatment is given after a prolonged dark incubation period at 20°C, secondary dormancy prevents germination. Factors controlling the induction and breaking of secondary dormancy have been investigated. The induction of secondary dormancy is very temperature dependent. Locally puncturing the seed coat strongly delays it. Secondary dormancy is not induced in the presence of GA₃ during the first 10 d of dark incubation, although this growth substance cannot induce dark germination. Prolonged or cyclic daily R irradiations can relieve secondary dormancy of seeds kept on water, even after a dark period of 20 d. A 24 h treatment at 4°C restores responsiveness to short R exposures of slightly secondarily dormant seeds. The synergism between GA₃ and Pfr in non-dormant Kalanchoë seeds, leading to high effectiveness of even one short FR irradiation, still occurs in seeds made secondarily dormant before transfer to GA₃, but more R or FR irradiations, in combination with GA₃, are required for the release of secondary dormancy. A combination of red light and 6-benzyl-aminopurine is ineffective in removing dormancy.     

Role of light in the regulation of the nitrate reductase level in wheat (Triticum aestivum)

The relationship between light and NR synthesis was studiedin excised wheat leaves. In light, there was a 14 fold increasein the NR level as compared to the dark controls. Sucrose didnot have any effect on NR synthesis. In the dark, a significantamount of intracellular nitrate was detected, but there wasvery little NR activity. Measurement of the decay rates of NRin the dark and light showed that NR activity disappeared fasterin light than in the dark. Exposure to short durations of blue,red and white light caused an increase in the NR synthesis.Further, this response was found to be phytochrome controlled.We propose that the process of nitrate reduction influencesNR synthesis. (Received January 11, 1979; )     

Effect of levulinic acid on pigment biosynthesis in Agmenellum quadruplicatum.

When levulinic acid was added to a growing culture of the cyanobacterium (blue-green alga) Agmenellum quadruplicatum PR-6, delta-aminoelevulinic acid accumulated in the medium and chlorophyll a synthesis and cell growth were inhibited, but there was a small amount of c-phycocyanin synthesis. The amount of delta-aminolevulinic acid produced in the treated culture did not fully account for the amount of pigment synthesized in the untreated control. Levulinic acid and either sodium nitrate or ammonium chloride were added to nitrogen-starved cultures of PR-6, and delta-aminolevulinic acid production and chlorophyll a and c-phycocyanin content were monitored. When ammonium chloride was added as a nitrogen source after nitrogen starvation, the cells recovered more rapidly than when sodium nitrate was added as a nitrogen source. In cultures recovering from nitrogen starvation, synthesis of c-phycocyanin occurred before synthesis of chlorophyll a.     

Photoreactivating enzyme from Escherichia coli: appearance of new absorption on binding to ultraviolet irradiated DNA.

The photoreactivating enzyme, PRE, monomerizes pyrimidine dimers in DNA in a light requiring reaction (lambda greater than 300 nm). However, the purified PRE from E. coli has no well-defined absorption band for lambda greater than 300 nm. Using absorption difference spectroscopy, we show that when PRE is mixed with ultraviolet-irradiated DNA, new absorption appears in the spectral region required for catalysis. There is a concomitant decrease in the absorption of the mixture for wavelength less than 300 nm. The hyperchromicity for lambda greater than 300 nm is true absorption, not an artifact due to light scattering. Both the hyperchromicity (lambda greater than 300 nm) and hypochromicity (lambda less than 300 nm) can be reversed by irradiation of 365 nm with identical first-order kinetics. We estimate the molar extinction coefficient of the new absorption to be 6900 +/- 1400 at 350 nm. We conclude that the PRE from E. coli does not possess a distinct "chromophore" which by itself is entirely responsible for the absorption of photoreactivating light. Instead, new absorption results when PRE binds its substrate, dimer-containing DNA.     

Action Spectrum for the Timing of Photo-Induced Cell Division in Adiantum Gametophytes

The photo-induced cell division in single-celled protonemata of the fern Adiantum capillus-veneris was studied. When the protonemata were exposed to monochromatic light at 50 nm intervals between 350 and 750 nm, irradiations in the blue and near-ultraviolet regions effectively induced cell division, while wavelengths longer than 550 nm showed no such effect. As reciprocity between duration and intensity was observed within the range of incident energy used, the action spectrum for the frequency of the photo-induced cell divisions 24 h after irradiation was determined between 360 and 510 nm at 10 nm intervals. Furthermore, the previously known effect of phytochrome on the timing of the cell division was minimized by a short exposure to red light given immediately after the monochromatic irradiation. The resulting action spectra showed a peak in the neighborhood of 460 nm with shoulders and another peak in the near-ultraviolet region.