Phytochrome-mediated inhibition of coleoptile growth in rice: age-dependency and action spectra

Phytochrome has been shown to be the major photoreceptor involved in the photo-inhibition of coleoptile growth in Japonica-type rice (Oryza sativa L.). We have characterized this typical photomorphogenetic response of rice using mutants deficient in phytochrome A (phyA) and phytochrome B (phyB) and with respect to age-dependency and action spectra. Seedlings were irradiated with a pulse of light 40 h or 80 h after germination (i.e. at an early or late developmental stage) and the final coleoptile length of these seedlings was determined. A saturating pulse of red light (R) had a stronger effect when it was given in the late stage than in the early stage. It was found that the photoinhibition is mediated by both the phyA and the phyB in the late stage but predominantly by phyB in the early stage. Consistent with many other reported responses, the photo-inhibition in the phyA mutant, which was observed in the early and late developmental stages and is thought to be mediated mainly by phyB, occurred in the low-fluence range (10(1)-10(3) micromol m(-2)) of R and was far-red-light (FR)-reversible; the photo-inhibition in the phyB mutant, which was observed in the late developmental stage and is thought to be mediated mainly by phyA, occurred in the very-low-fluence range (10(-2)-10(0) micromol m(-2)) and was FR-irreversible. The action spectra (350-800 nm at 50 nm intervals) obtained at the two developmental stages using phyA and phyB mutants indicated that both the phyB-mediated low-fluence response and the phyA-mediated very-low-fluence response have a major peak at 650 nm and a minor peak at 400 nm.     

ACTION SPECTRA IN ULTRAVIOLET WAVELENGTHS (150-250 nm) FOR INACTIVATION AND MUTAGENESIS OF Bacillus subtilis SPORES OBTAINED WITH SYNCHROTRON RADIATION

Bacillus subtilis spores were exposed in vacuo to monochromatic UV radiation from synchrotron radiation in the wavelength range of 150 nm to 250 nm. Survival and frequency of mutation to histidine-independent reversion were analysed for three types of spores differing in DNA-repair capabilities. UVR spores (wild-type DNA repair capability) exhibited nearly equal sensitivity to the lethal effects of far-UV (220 nm and 250 nm) and of vacuum-UV radiation (150 and 165 nm), but showed marked resistance to 190 nm radiation. UVS spores (excision-repair and spore-repair deficient) and UVP spores (a DNA polymerase I-defective derivative of UVS) exhibited similar action spectra; pronounced sensitivity at 250 and 220 nm, insensitivity at 190 nm and a gradual increase of the sensitivity as the wavelength decreased to 165 nm. In all strains, the action spectra for mutation induction paralleled those for the inactivation, indicating that vacuum-UV radiation induced lethal and mutagenic damages in the spore DNA. The insensitivity of the spores to wavelengths around 190 nm may be explicable by assuming that radiation is absorbed by materials surrounding the core in which DNA is situated.     

INACTIVATION ACTION SPECTRA OF BACILLUS SUBTILIS SPORES IN EXTENDED ULTRAVIOLET WAVELENGTHS (50–300nm) OBTAINED WITH SYNCHROTRON RADIATION

Five types of Bacillus subtilis spores (UVR, UVS, UVP, RCE, and RCF) differing in repair and/or recombinational capabilities were exposed to monochromatic radiations at 13 wavelengths from 50 to 300 nm in vacuum. An improved biological irradiation system connected to a synchrotron radiation source was used to produce monochromatic UV radiation in this extended wavelength range with sufficient fluence to inactivate bacterial spores. From the survival curves obtained, the action spectra for the inactivation of the spores were depicted. Recombination-deficient RCE (recE) and RCF (recF) spores were more sensitive than the wild-type UVR spores in the entire range of wavelengths. This was considered to mean that DNA was the major target for the inactivation of the spores. Vacuum-UV radiations of 125-175 nm were effective in killing the spores, and distinct peaks of the sensitivity were seen with all types of the spores. Insensitivities at 190 and 100 nm were common to all five types of spores, indicating that these wavelengths were particularly impenetrant and absorbed by the outer layer materials. The vacuum-UV peaks centering at 150 nm were prominent in the spores defective in recombinational repair, while the far-UV peaks at around 235 and 270 nm were prominent in the UVS (uvrA ssp) and UVP (uvrA ssp polA) spores deficient in removal mechanisms of spore photoproducts. Thus, the profiles of the action spectra were explained by three factors; the penetration depth of each radiation in a spore, the efficiency of producing DNA damage that could cause inactivation, and the repair capacity of each type of spore.     

PHOTOMORPHOGENIC RESPONSES TO UV RADIATION. II: A COMPARATIVE STUDY OF UV EFFECTS ON HYPOCOTYL ELONGATION IN A WILD-TYPE AND AN aurea MUTANT OF TOMATO (Lycopersicon esculentum Mill.)

Hypocotyl growth in etiolated seedlings of wild-type and an aurea mutant of tomato (Lycopersicon esculenturn Mill.), that appears to be deficient in labile phytochrome, is strongly inhibited by UV radiation in the region of 300–400 nm. The role of phytochrome in the UV-mediated inhibition of hypocotyl growth was studied using different experimental approaches: (1) by comparing the effectiveness of treatments of increasing duration of exposure to 692 nm and UV radiation; (2) by modifying the UV spectral range with specific cut-off filters. The experimental results suggest that the UV-induced inhibition of growth in wild-type tomato is mediated to a large extent by the longer wavelengths of the UV-A region and is mediated mainly by phytochrome. In contrast, at wavelengths < 305 nm a strong UV-B effect was found in the aurea mutant, suggesting a preeminent action of a specific UV-B absorbing photoreceptor that displays less action in the wild-type.     

Blue and red light-induced germination of resting spores in the red-tide diatom Leptocylindrus danicus

Photophysiological and pharmacological approaches were used to examine light-induced germination of resting spores in the red-tide diatom Leptocylindrus danicus. The equal-quantum action spectrum for photogermination had peaks at about 440 nm (blue light) and 680 nm (red light), which matched the absorption spectrum of the resting spore chloroplast, as well as photosynthetic action spectra reported for other diatoms. DCMU, an inhibitor of photosynthetic electron flow near photosystem II, completely blocked photogermination. These results suggest that the photosynthetic system is involved in the photoreception process of light-induced germination. Results of pharmacological studies of the downstream signal transduction pathway suggested that Ca(2+) influx is the closest downstream neighbor, followed by steps involving calmodulin, nitric oxide synthase, guanylyl cyclase, protein-tyrosine-phosphatase, protein kinase C and actin polymerization and translation.     

ACTION SPECTRA FOR INHIBITION OF HYPOCOTYL GROWTH OF WILD-TYPE PLANTS AND OF THE hy2 LONG-HYPOCOTYL MUTANT OF Arabidopsis thaliana L.

Abstract— An analysis was made by action spectroscopy, using the Okazaki Large Spectrograph, of the inhibition of hypocotyl elongation of wild-type plants and the hy2 mutant of Arabidopsis thaliana. Two day old etiolated seedlings were irradiated for 8 h with monochromatic light and left in the dark for 16 h before measurement of hypocotyl length. Spectrophotometric measurement showed that levels of phytochrome in the etiolated tissue of the hy2 mutant were less than 9% of those in the wild type. The action spectra of the wild type looked like those of high irradiance response and showed peaks at 375, 450, 625 and 725 nm, whereas the action spectra of hy2 showed only the peaks at 375 and 450 nm. Monochromatic light of wavelengths longer than 500 nm had no significant inhibitory effects on hy2 plants. Blue and UV-A light were about five times more effective in the wild type than in hy2 plants. Severe inhibitory effects were observed with UV-B light. It is concluded that inhibition of the growth of the hypocotyl involves combined actions of phytochrome and a putative blue/UV-A photoreceptor(s).     

AN ACTION SPECTRUM FOR ULTRAVIOLET-INDUCED SPORULATION IN THE FUNGUS STEMPHYLIUM SOLANI WEBER

Abstract— An action spectrum for conidial (spore) production in the fungus Stemphylium solani Weber is presented. The response is in the far-ultraviolet region (far u.v.). The spectrum shows a single peak at 280 nm, a shoulder at 260 nm, a trough at 250 nm, followed by a rise which is still rising at 230 nm, the shortest wavelength studied. The u.v. induced spore stimulation can be interrupted temporarily by irradiation in the visible region within 4 hr after induction.     

PHOTOMORPHOGENIC RESPONSES TO UV RADIATION III: A COMPARATIVE STUDY OF UVB EFFECTS ON ANTHOCYANIN and FLAVONOID ACCUMULATION IN WILD-TYPE and aurea MUTANT OF TOMATO (Lycopersicon esculentum MILL.)

The UV-mediated induction of anthocyanin and UV-absorbing compounds was characterized in etiolated hypocotyls of wild-type and aurea (au) mutant tomato seedlings. Ultraviolet radiation induced significant increases of anthocyanin and UV-absorbing compounds in hypocotyls of die au mutant and of its isogenic wild-type, but the differences in the time courses of UV-induced pigment accumulation indicate mat different photoregulatory mechanisms are involved for each of these two groups of pigments. It appears mat prolonged presence of adequate levels of UVB (290–320nm) energy and consequently the action of a specific UVB photoreceptor are indispensable for the photoinduction of anthocyanin accumulation in UV-irradiated hypocotyl of the au mutant that is missing the labile phytochrome pool. The large difference found between the wild-type and the au mutant strongly indicate the involvement of labile phytochrome as the primary functional photoreceptor for the photoinduction of anthocyanin accumulation in wild-type tomato hypocotyls. The UVB photoreceptor could at least partly replace the action of labile phytochrome (as far as anthocyanin accumulation is concerned) when the functional phytochrome pool is missing as in the au mutant. The general picture of UV-mediated induction of total UV-absorbing compounds shows only a macroscopic difference between wild-type and die au mutant of tomato: the higher initial level (in darkness) of these compounds in die wild-type in contrast to the au mutant. Although there is UV-induced accumulation of UV-absorbing compounds in bom genotypes, the levels in the au mutant never reach mat of the wild-type under the same UV exposure. A UVB photosensor may play a more important role in the photoinduction of UV-absorbing compounds. Indeed, in the absence of labile phytochrome, i.e. in the au mutant, a UVB-absorbing photoreceptor alone is able to establish high responsiveness for the UV-induced flavonoid accumulation.     

STUDIES ON THE PROTEIN CONFORMATION OF PHYTOCHROME

Abstract— The extinction coefficients for large rye phytochrome were found to be: Fluorescence and circular dichroism spectra of large- and small-molecular-weight rye phytochrome give no evidence for a protein conformational change on phototransformation of phytochrome. The large molecule has a fluorescence emission peak at 331 nm when excited at 290 nm, and an excitation peak for this emission at 288 nm. The circular dichroism spectra indicate that large rye phytochrome has about 17–20% a-helix content, 30%β-structure and 50% random coil, and that the small rye phytochrome has about 10–13%α-helix content. The ultraviolet difference spectra for large and small rye phytochrome are similar and differ from the difference spectrum of the small oat phytochrome in the relative size of the 296–298 nm peak. The difference spectra may reflect changes in chromophore absorbance and in the environment of amino acid residues near the chromophore, particularly of tyrosine, and perhaps of tryptophan and cysteine.     

Comparisons of spore dosimetry and spectral photometry of solar-UV radiation at four sites in Japan and Europe

In order to develop monitoring and assessment systems of biologically effective doses of solar-UV radiation, concurrent measurements of spectral photometry and spore dosimetry were conducted in summer months at four sites in Japan and Europe. Effectiveness spectra were derived by multiplying spectral irradiance in 0.5 nm steps between 290 and 400 nm with the inactivation efficiency of the spores determined using monochromatic radiation of fine wavelength resolution. Shapes of the effectiveness spectra were very similar at the four sites exhibiting major peaks at 303.5, 305.0, 307.5 and 311.0 nm. The dose rates for spore inactivation from direct survival measurements and from calculations by the integration of the effectiveness spectra were compared for 174 data points. The ratios (observed/calculated) of the two values were concordant with a mean of 1.26 (+/- 0.24 standard deviation [SD]). The possible causes for the variations and slightly larger observed values are discussed.     

MODIFIED ACTION SPECTRA OF PHOTOGEOTROPIC EQUILIBRIUM IN Phycomyces blakesleeanus MUTANTS WITH DEFECTS IN GENES madA, madB, madC, and madH

Abstract— Action spectra of photogeotropic equilibrium were measured for behavioral mutants of Phycomyces blakesleeanus with defects in the genes madB, madC and madH as well as for a double mutant defective in the genes madA and madC. The action spectra of strains C109 (madB), LI (madC) and L52 (madA madC) all lack the broad near-ultraviolet peak which extends from 347 to 386 nm in the wild type; the peaks at 414 and 491 nm are also missing in these mutants. The double mutant L52 (madA madC) shows a novel broad peak at 477 nm; the relative quantum effectiveness of L52 at 477 nm is 10 times higher than in LI (madC119). These properties of the double mutant L52 (madA madC) suggest steric interaction of the madA and the madC gene products in the photoreceptor complex. For the hypertropic mutant L84 (madH) the action spectrum and absolute sensitivity are similar to those for wild type. These results confirm and extend previous findings that multiple photoreceptors are mediating phototropism in P. blakesleeanus.     

SOLAR DOSIMETRY WITH REPAIR DEFICIENT BACTERIAL SPORES: ACTION SPECTRA, PHOTOPRODUCT MEASUREMENTS AND A COMPARISON WITH OTHER BIOLOGICAL SYSTEMS

Abstract— Populations of radiation sensitive spores ( Bacillus subtilis UVSSP), vegetative bacteria ( E. coli K12-AB2480) and bacteriophage ( E. coli phage T4vx) have been considered as possible biological dosimeters to integrate DNA-absorbed solar energy incident on the Earth's surface.
Irradiation of spores of B. subtilis UVSSP with monochromatic far- and near-UV radiation and solar radiation have indicated that these radiations have a similar efficiency in inducing spore photoproducts per lethal event. Action spectra for lethality taken with the three radiation sensitive biological systems show a similar pattern in each case with a broad shoulder in the 334–365 nm wavelength region. This finding indicates a relatively high susceptibility of the DNA to chemical alteration in this wavelength range. Although less sensitive to sunlight than the other biological systems tested, the B. subtilis UVSSP spore mutant has the advantage of temperature independence of inactivation, stability between irradiation and assay and a simple, reproducible irradiation and assay procedure. Field measurements have supported the utility of this mutant as a sunlight dosimeter.     

MULTIPLE EFFECTS OF UV RADIATION (265–330 NM) ON FUNGAL SPORE EMERGENCE

We have investigated the influence of narrow-band UV radiation, 265–330 nm. on germination of spores of the fungus Cladosporium cucumerinum Ellis and Arth., using a Xe arc lamp and filters. Reciprocity of time and dose rate was demonstrated when fungal spores were subjected to UV radiation at 325 nm but failed to hold at 265 nm. Based on these findings, data on fluence response, and partial action spectra, we propose that there are two biologically active sites in this organism that are affected by radiation between 265 and 330 nm and that might be influenced by changes in the stratospheric ozone layer: a short-wave-sensitive site (265–295 nm) and a long-wave-sensitive site (300–330 nm). Data obtained with narrow-band interference filters confirmed previous reports of damage to nucleic acid from UV at 265–295 nm and in addition demonstrated significant inhibition by UV at 300–320 nm. Further studies of the 300 330 nm portion of the spectrum, using combinations of plastic and glass filters, showed that the influence of UV radiation in this region was primarily to produce a non-photoreactivable delay in germ-tube emergence.     

Equal-quantum action spectra indicate fluence-rate-selective action of multiple photoreceptors for photomovement of the thermophilic cyanobacterium Synechococcus elongatus

Unicellular thermophilic cyanobacterium Synechococcus elongatus displayed phototaxis on agar plate at 55 degrees C. Equal-quantum action spectra for phototactic migration were determined at various fluence rates using the Okazaki Large Spectrograph as the light source. The shapes of the action spectra drastically changed depending on the fluence rate of the unilateral monochromatic irradiation: at a low fluence rate (3 mumol/m2/s), only lights in the red region had significant effect; at a medium fluence rate (10 mumol/m2/s), four major action peaks were observed at 530 nm (green), 570 nm (yellow), 640 nm (red) and 680 nm (red). At high fluence rates (30-90 mumol/m2/s), the former two peaks remained, while red peaks at 640 nm and 680 nm disappeared and, interestingly, an action peak around 700-740 nm (far-red) newly appeared. These results indicate that two or more distinct photoreceptors are involved in the phototaxis and that suitable photoreceptors are selectively active in response to the stimulus of light fluence rates. Far-red or red background lights irradiated vertically from above drastically inhibited phototaxis toward red light or far-red light, respectively. These results indicate involvement of some phytochrome(s).     

SITE-DIRECTED MUTAGENESIS OF THE LH2 LIGHT-HARVESTING COMPLEX OF Rhodobacter sphaeroides: CHANGING βLys23 TO Gin RESULTS IN A SHIFT IN THE 850 nm ABSORPTION PEAK

The present study describes the construction of a Rhodobacter sphaeroides light-harvesting (LH2) mutant in which the charged residue βSLys23 is changed by site-directed mutagenesis to a Gin residue, and the characterization of the resulting mutant complex by a range of spectroscopic techniques. In the 77 K absorption spectrum of the mutant, the peak equivalent to the 850 nm peak in the wild-type membrane is blue-shifted by approximately 18 nm to 837 nm; except for this blue-shift, the 77 K. fluorescence excitation and emission spectra and the circular dichroism spectrum of the mutant are very similar to the equivalent spectra from the wild-type membranes, suggesting that the mutation βLys23 → Gin probably does not cause any major changes in the conformation or aggregation state of these membranes. Possible causes of the 18 nm blue-shift in the absorption spectrum are discussed.     

Photoreactivation of UV-lnactivated Spores of Trichoderma harzianum

Abstract— Photoreactivation in the filamentous soil fungus Trichoderma harzianum is of interest because its blue, UVA photoreceptors (cryptochromes) may share homology with DNA photolyases. Furthermore, this organism antagonizes, by mycoparasitism, a number of soil-borne pathogens. Photoreactivation is thus important as one of the factors that may contribute to survival in the field. Exposure of asexually produced spores (conidia) to UVC inhibits germination. Nongerminating spores either do not swell or are inhibited later in germination, swelling but failing to put out a germ tube. Both types of inhibition can be reversed by photoreactivation with visible and UVA (320-400 nm) light, restoring high germination percentages. Conidia of mutants lacking the normal greenish pigmentation are more sensitive to UVC (200-280 nm) than wild-type conidia but photoreactivation still occurs. The action spectrum for photoreactivation indicates that T. harzianum has a DNA photolyase with a pterin as second chromophore. The most effective wavelengths for photoreactivation correspond to valleys, rather than peaks, in the action spectrum for photoinduction of sporulation. Furthermore, mutants with defects in photoinduction of sporulation ( dimY ) are not defective in photoreactivation. Induction of sporulation and DNA photorepair, while sharing parts of the blue/UVA spectrum, are different, by spectroscopic, kinetic and genetic criteria.     

LIGHT-INDUCED SYNTHESIS OF ANTHOCYANIN IN CARROT CELLS IN SUSPENSION—IV. THE ACTION SPECTRUM

Abstract— Using carrot cell suspension in 2,4-dichlorophenoxyacetic acid (2,4-D)-depleted culture medium, fluence-response curves for the formation of anthocyanin were determined at various wavelengths from 250 to 800 nm. In the fluence-response curves at wavelengths between 260 and 330 nm, the response showed a sharp fluence-dependent increase after the fluence exceeded threshold level at the respective wavelength. Such a sharp increase in response was not observed by light at 450 nm or longer wavelengths, although the response obtained by higher fluence of such light was always higher than that in the dark control. Action spectra determined at the sharp increasing phase of the response showed the single peak at 280 nm which equals the absorption maximum of UV-B photoreceptor.
Although red (R)-light alone had a minor effect on anthocyanin accumulation, it modulated the action of UV-B light. That is, when carrot cells were irradiated with R-light either before or after UV-B irradiation, anthocyanin formation was greatly enhanced above the level enhanced by UV-B light alone. The most effective wavelength for this enhancement was 660 nm. The effect of R-light on the anthocyanin formation of the UV-B irradiated cells was reversed by immediately following it with far-red light, suggesting the involvement of phytochrome in the R-effect.     

LIGHT INDUCED FLUORESCENCE SPECTRAL CHANGES IN NATIVE PHYTOCHROME FROM Secale cereale L. AT LIQUID NITROGEN TEMPERATURE

Abstract— Fluorescence spectra of native rye phytochrome were determined under different light conditions at liquid nitrogen temperature. Fluorescence spectrum of the red-light-absorbing form (Pr) had a major peak at about 685 nm (14 600 cm⁻¹) and a broad sub-peak at about 515 nm (19 400 cm⁻¹). The peak height at 685 nm was reduced by irradiation with monochromatic light of 640 nm, and a new peak became obvious at about 702 nm (14250 cm⁻¹). This spectral change was almost completely reversed by subsequent irradiation with 700-nm light. Fluorescence spectrum of the photoequilibrium mixture of Pr and far-red-light absorbing form under continuous red light showed a sharp peak at about 685 nm having a peak height ca. 12% of Pr, and a broad sub-peak at about 508 nm (19 700 cm⁻¹). Light of 730 nm did not reduce the peak height at about 685 nm but induced a new shoulder at about 699 nm (14300 cm⁻¹). Monochromatic light of 640 and 700 nm given following the light of 730 nm could not reverse the spectral change at 699 nm induced by the irradiation with 730-nm light. Fluorescence spectrum of Pr in partially degraded phytochrome was similar to that in native phytochrome but the peak position in the red region was shifted by about 5 nm (100 cm⁻¹) to the blue.     

THE USE OF A COMPUTERIZED SPECTRORADIOMETER TO PREDICT PHYTOCHROME PHOTOEQUILIBRIA UNDER POLYCHROMATIC IRRADIATION

Abstract A method is described for predicting the effect of polychromatic irradiation upon the photo-stationary equilibrium of the plant photoreceptor phytochrome. This method follows from the rate equations for phototransformation and utilizes the in vivo action spectra for phytochrome phototransformation (Pratt and Briggs, 1966). A scanning spectroradiometer interfaced with a microcomputer is used to determine a spectral photon distribution from 360 to 800 nm. The products of the photon fluence rate and the relative quantum efficiencies at 2-nm intervals are summed over the entire visible range to yield a predicted percentage of the pigment in the Pfr form. This value was determined under eight different polychromatic light sources and was generally within 7% Pfr of the value measured in etiolated corn coleoptiles under the same light sources.     

ACTION SPECTRUM FOR PHOTOREVERSION OF THE ACTIVE Pfr-FRACTION OF Mougeotia in vivo

Abstract— The dichroic oriented fraction of the far-red light absorbing form of phytochrome (Pfr) in the green alga Mougeotia was characterized by action spectroscopy. Microbeam irradiations had to be used for the induction of chloroplast movement in Pfr-containing cells, because of the special dichroic absorption characteristics of the red light absorbing form of phytochrome (Pr) and Pfr in the alga. Fluence-response curves were elaborated especially in the far-red spectral region by reverting Pfr to Pr at the flanks of the cells and thus generating Pfr-gradients. Linearly polarized light vibrating perpendicularly to the cell axis was used, thus corresponding to the S,-transition moments of Pfr at the flanks of the cells. The action spectrum is characterized by a peak at approximately 715 nm and a very pronounced decrease towards 728 and 734 nm. The data indicate that the spectral absorption of the active Pfr-fraction in green Mougeotia is shifted towards shorter wavelengths as compared to extracted phytochrome from etiolated or even green higher plants. This "blue shift" seems to be typical for Pfr from green lower plants.