AN ACTION SPECTRUM IN THE BLUE and ULTRAVIOLET FOR PHOTOTROPISM IN ALFALFA*

Abstract Phototropism is a common property of plants, but it is not known if different species use the same photoreceptor for their response. We have determined fluence-response relations for phototropism in response to brief, broad-band blue irradiation for four plant species grown under red light (Amaranthus paniculatus, Linum usitatissimum, Vigna radiata and Medicago sativa) and compared these to ones previously obtained for Pisum sativum and Zea mays, grown under similar conditions. Curves for all species showed a bell-shaped dependence on fluence, a characteristic of first positive curvature as originally defined for the Avena coleoptile, and had a similar optimal fluence, near 3 H.mol m^?2. We have obtained an action spectrum in the blue and UV spectral regions for first positive phototropism of the hypocotyl of alfalfa grown under red light. Fluence-response curves at wavelengths between 300 and 500 nm were nearly identical in shape and magnitude; whereas below 300 nm, their slopes and maximum curvatures were reduced. The action spectrum showed that activity rose sharply at wavelengths below 500 nm, peaked at 450 nm with shoulders on either side of that peak, and had lesser peaks at 380 and, in the far ultraviolet, at 280 nm. This action spectrum was very similar to ones in the literature (obtained between 350 and 500 nm) for first and second positive phototropism of oat coleoptiles. We conclude that the same photoreceptor mediates phototropism in oat and alfalfa.     

Biphasic fluence-response curves and derived action spectra for light-induced absorbance changes in Phycomyces mycelium

The photoresponses of Phycomyces, including phototropism and photocontrol of sporangiophore development, are mediated primarily by blue and UV light. Recent results on these two responses indicated a subsidiary role for green light. We have measured in vivo light-induced absorbance changes (LIAC) in mycelial samples of a caroteneless (carB) strain to compare the effectiveness of UV, blue, and green light. In the dual-wavelength kinetic mode of the spectrophotometer, measuring wavelengths of 445 and 470 nm were chosen, because green light produces substantial absorbance changes between these two wavelengths. Fluence-response curves were measured for 13 wavelengths between 365 and 530 nm, and for variable exposure times between 0.5 and 320 s. With one exception (365 nm), the curves were biphasic. The low fluence component was generally sigmoidal with an abrupt rise. The high fluence component failed to reach saturation for the fluences tested (less than 70 μmol m⁻² s⁻¹). Using the inferred threshold fluences of these two components as criterion effects, we obtained two action spectra. For the low fluence component, the action spectrum showed major peaks at 394, 450, and 530 nm and a minor peak at 416 nm. The high fluence component action spectrum showed very little sensitivity in the blue region. The major sensitivity was in the near UV, and a relatively small peak appeared in the green part of the spectrum at 507 nm. The biphasic character of the fluence-response curves suggests that two photosystems are responsible for the absorbance changes. The low fluence photosystem is sensitive mainly to blue and UV light and may thus represent a physiological blue-light photoreceptor. The high fluence photosystem is clearly not of this type. It (and perhaps the low fluence system as well) may mediate some of the subsidiary physiological effects of green light.     

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.     

UVB Monochromatic Action Spectrum for the Inhibition of Photosynthetic Oxygen Production in the Green Alga Dunaliella saha

The effect of monochromatic UVB (280–315 nm) irradiation on the photosynthetic activity of the marine green alga Dunaliella salina was investigated by monitoring the rate of oxygen exchange. Samples were irradiated with narrow bands centered at different UV wavelengths (281, 290, 300, 310 and 322 nm). In a first set of experiments the samples were preirradiated keeping constant the irradiance and varying exposure time. By increasing UVB fluence, a wavelength-dependent decrease in the rate of oxygen production was observed. In a second set of experiments oxygen exchange was monitored simultaneously to UVB irradiation. The decline in the rate of oxygen production was sharper at short wavelengths and faster with increasing UVB photon flux density. The photon fluence action spectra derived from the two sets of experiments are in good agreement and indicate a decrease in the UVB-inhibiting effectiveness of two orders of magnitude from about 0.2 (mmol m-2)-1 at 281 nm to about 0.003 (mmol m-2)-1 at 322 nm. The photon flux density action spectrum derived from the second set of experiments indicates a smoother decrease from about 0.2 (pmol s-l m-2)-1 at 281 nm to about 0.05 (pmol s-l m-z)-l at 310 nm.     

ACTION SPECTRA FOR PHOTOTROPIC BALANCE IN Phycomyces blakesleeanus: DEPENDENCE ON REFERENCE WAVELENGTH and INTENSITY RANGE

Abstract— Action spectra for phototropic balance of Phycomyces blakesleeanus sporangiophores were measured for various reference wavelengths and intensity ranges. Balance action spectra were made at fluence rates of 10^-4 W m^-2 with reference wavelengths of 450 nm, 394 nm, 507 nm, and broadband blue light. For broad-blue light and 450 nm light as references, typical flavin-like action spectra were found with a ma jor peak at 455 nm, a secondary peak at 477 nm, and a minor peak at 383 nm; these peaks are wider for broad blue than for 450 nm light. With the 394 nm reference, there is a major peak at 455 nm, a secondary peak at 477 nm and a minor peak at 394 nm. An action spectrum with 507 nm reference has a major peak at 455 nm and a minor peak at 383 nm, but no peak at 477 nm. A balance action spectrum was made with 450 nm reference light near threshold intensity (2 times 10^-8 W m^-2); there, the 386 nm peak is greatly reduced, while the 455 nm peak is enhanced. The intensity dependence of the 386 nm peak was studied in detail for reference light of 450 nm. We found that the relative quantum efficiency of the 386 nm light increases with the logarithm of the 450 nm fluence rate; in the high intensity range (0.3 W m^-2) the relative quantum efficiency of the 386 nm light is 1.3 and approaches zero at 10^-9 W m^-2. These findings indicate that P. blakesleeanus phototropism is mediated by multiple interacting pigments or by a photochromic photoreceptor.     

ACTION SPECTRUM FOR SUBLIMINAL LIGHT CONTROL OF ADAPTATION IN Phycomyces PHOTOTROPISM

Abstract -Adaptation processes enable phototropism and other blue light responses of Phycomyces to operate over a 10-decade range of Ruencc rate. Phototropic latency, used routinely to monitor the kinetics of sensitivity recovery after a step down in fluence rate, can be shortened by application of dim light for 35 min during the early part of the latency period. This light is termed subliminal , because it does not elicit phototropism under these experimental conditions; rather, it exerts its influence on the underlying adaptation kinetics. Fluence rate-response data for this latency reduction, obtained at 17 wavelengths of subliminal light from 347 to 742 nm, showed a variety of shapes that could be fit by zero, one, or two sigmoidal components, plus a constant term. At most wavelengths, the fluence-rate threshold for latency reduction by subliminal light tended to be well below the absolute threshold for phototropism, indicating that this effect is highly sensitive. An action spectrum for the sensitivity of the subliminal light effect, derived from the fluence rate-response curves, shows major peaks around 400 and 500 nm and a broad band from 570 to 670 nm, followed by a steep absorption edge. The sensitivity in the near ultraviolet region is relatively very low. The magnitude of the latency reduction also depends strongly on wavelength with a maximum at about 450 nm. The Huence-rate response data and the action spectrum–which is markedly different from that for phototropism and other blue-light responses of Phycornyces – indicate the participation of multiple pigments, or pigment states, in the photocontrol of adaptation.     

Photoreception and Phototropism in Phycomyces: Antagonistic Interactions between Far-UV, Blue, and Red Light

Abstract— Phototropism of the sporangiophore of the fungus Phycomyces is mediated by UV and blue light. Classical phototropism action spectra with maxima near 280, 370 and 450 nm indicate a flavin-like photoreceptor. Blue light mediates positive phototropism while far-UV light mediates negative phototropism. To better understand the mode of interaction of far-UV with blue light we performed phototropism experiments in which sporangio-phores were placed for 4 h between sources of 280 and 454 nm light coming from opposite directions. The fluence rates of the far-UV were chosen such that unilateral light alone elicited 90° of negative bending. For blue light, moderate fluence rates were applied that elicited about 40° bending. Under conditions of bilateral irradiation the blue light substantially reduced the far-UV elicited phototropism. In the presence of tonic red light the antagonism between far-UV and blue light was greatly reduced. Red light, which by itself is phototropically ineffective, also reduced phototropic bending elicited by either far-UV or blue light. These observations are taken as indications for the existence of a red light-absorbing intermediate of the blue-light receptor. Because the far-UV/ blue-light antagonism disappeared almost completely in the presence of tonic red light, the antagonism may occur at the level of this receptor intermediate.     

ACTION SPECTRA FOR SUPEROXIDE GENERATION AND UV AND VISIBLE LIGHT PHOTOAVOIDANCE IN PLASMODIA OF Physarum polycephalum

Abstract— The initial photochemical process leading to photoavoidance by plasmodia of an albino strain of Physarum Plasmodium was studied. Superoxide (O), detected as superoxide dismutase (SOD)-inhibitable electron spin resonance (ESR) signal of a spin trap (tBN), was formed upon irradiation. The amount of O formed increased linearl) with log fluence rate above the threshold. The photoavoidance to radiation at wavelengths between200–800 nm also showed the similar linear relationship in log fluence rate-response curves. Thresholds for photoavoidance and O generation agreed with each other and the action spectra showed peaks at about 260, 370, and 460 nm. Thus, active oxygen generated by photosensitization seems to trigger the UV and blue light photoavoidance.     

A PHOTORECEPTOR SYSTEM REGULATING in vivo AND in vitro PHOSPHORYLATION OF A PEA PLASMA MEMBRANE PROTEIN*

Abstract— We have continued to characterize the blue light-regulated phosphorylation of a 120 kDa pea plasma membrane protein thought to be involved in sensory transduction for phototropism (Short and Briggs, 1990, Plant Physiol. 92 , 179–185). By incubating pea stem sections in ³²P-phosphate, we show that the 120 kDa protein is phosphorylated in vivo only after blue light irradiation and that the photosensitive fluence range matches that for phototropism. Blue light induces phosphorylation of the protein in vitro as well, but the fluences required to elicit the response are at least 30-fold higher. Triton solubilization of the plasma membrane vesicles does not further alter the fluence-response relationship. Very little turnover was detected over 20 min phosphorylation time courses or by pulse-chase experiments on unirradiated, blue light pulse-irradiated, or continuously irradiated membranes. Experiments with a dark period intervening between irradiation and addition of adenosine triphosphate show the light-induced change to persist for several minutes at 30°c. Agents that disrupt the normal photochemistry of flavins preferentially inhibit the light-induced enhancement of phosphorylation, suggesting a flavin chromophore. However, exogenous free flavins do not affect the sensitivity of the response. Staphylococcus aureus V-8 proteolysis of the phosphorylated protein from membranes subjected to a range of fluences before phosphorylation shows that the radiolabel on each of three peptides increases in proportion to the phosphorylation level of the undigested polypeptide. These studies may be valuable for assessing the nature of the photoreceptor and for unravelling the early sensory transduction steps in phototropism.     

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).     

WAVELENGTH DEPENDENCE (150–290 nm) OF THE FORMATION OF THE CYCLOBUTANE DIMER AND THE (6–4) PHOTOPRODUCT OF THYMINE

The action cross sections for the formation of the cyclobutane dimer and the (6-4) photoproduct of thymine as well as the absorption cross sections of thymine were determined in the wavelength region between 150 and 290 nm. Thymine films sublimed on glass plates were irradiated by monochromatic photons in a vacuum; the induced photoproducts were quantitatively analyzed by high-performance liquid chromatography (HPLC). Under our conditions, two major peaks appeared on the HPLC chromatograms of irradiated samples. The two peaks were identified as being the cis-syn cyclobutane dimer and the (6-4) photoproduct, based on their HPLC retention times, absorption spectra in the effluent, and photochemical reactivity. The fractions of the two photoproducts increased linearly with the fluence at low fluences over the entire wavelength range. Their action cross sections were determined by the slopes of the linear fluence response curve at 10 nm intervals between 150 and 290 nm. The two action spectra showed a similar wavelength dependence and had a maximum at 270 nm as well as two minor peaks at 180 and 220 nm, at which wavelengths the peaks of the absorption spectrum of thymine sublimed on a CaF2 crystal plate appeared. The quantum yields had relatively constant values of around 0.008 for the dimer and 0.013 for the (6-4) photoproduct above 200 nm, decreasing to 0.003 and 0.006, respectively, at 150 nm as the wavelength became shorter.     

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.     

Action Spectra for Photoinhibition of Sexual Development in Phycomyces blakesleeanus

The sexual development of the fungus Phycomyces is inhibited by light. The action spectra for this photoinhibitory effect were determined for 48 h continuous exposure between 350 and 700 nm wavelengths during the mating process. Effective wavelengths were shorter than 490 nm, but the most effective wavelengths depended on the stage of sexual development. In early stages of progametangium formation, the major peaks appeared near 360 nm with small shoulders at 410 nm, but in later stages, after gametangium formation, only single peaks were detected in the UVA range (350–390 nm). Low-fluence irradiation in the later stage, however, revealed inhibitory effectiveness at 370–410 nm, implying the existence of a dual photoresponse and multiple regulatiory systems in the mating process of Phycomyces.     

Comparison of action spectra for acute cutaneous responses to ultraviolet radiation: man and albino hairless mouse

Abstract The hairless mouse has been used as an experimental model for photocarcinogenesis for about 20 years. Although the carcinogenesis action spectra for mice and man are not known, acute responses to ultraviolet radiation (UVR) in the biologically active UVB and UVC region (wavelengths below 320 nm) can be compared. Vascular response (predominantly edema) action spectra for monochromatic radiation in the Skh:HR-l (albino hairless) male mouse were determined. These action spectra were found to be very similar to the human erythema action spectrum that had been developed using the same monochromator. The accuracy of this experimentally derived action spectrum was tested with a series of polychromatic source spectra. The mice were exposed to radiation from a long arc Xe lamp filtered by varying thicknesses of Schott WG320 filters, which yielded a wide range of biologically effective spectra. Spectral irradiance measurements, when weighted with the mouse edema and human erythema action spectra and multiplied by the irradiation time required to elicit a threshold response (edema), yielded a constant weighted dose regardless of irradiation spectral quality. The integrated effective dose was approximately 200 J/m² of 297 nm equivalent energy, agreeing with requirements for the monochromatic 297 nm dose in the mice as well as for minimal human erythema. These data suggest a commonality in the UVR chromophores of mice and men as they relate to the acute responses described, and a direct additivity of effectiveness from the UVR components in a polychromatic beam, at least over the portion of the UVR spectrum tested (λ > 295 nm).     

Ultraviolet action spectra for DNA dimer induction, lethality, and mutagenesis in Escherichia coli with emphasis on the UVB region

Abstract —Ultraviolet (UV) action spectra were obtained for lethality and mutagenesis (reversion to tryptophan independence) in Escherichia coli WP2s for wavelengths 254–405 nm with detailed analysis in the UVB region (290–320 nm). Parallel chemical assay yields of pyrimidine dimers in DNA of E. coli RT4 were determined at the same wavelengths. Spectral regions isolated from a Xe arc and resonance lines from a high-pressure Hg-Xe arc lamp were both used for irradiation. In all cases, precise energy distributions throughout the isolated Xe bands regions were defined.
Lethality, mutagenesis, and dimer induction all decreased in efficiency in a similar fashion as the wavelengths of the radiation increased. Between 300 and 320 nm, all characteristics measured showed differences of about two and a half orders of magnitude. Between these wavelengths, the values of the three end points used either coincide with or parallel the absorption spectrum of DNA. The mutagenesis action spectrum coincides closely with the absorption spectrum of DNA. The lethality spectrum is closely parallel to the mutagenicity spectrum; the points, however, consistently occur at about 2 nm longer wavelengths. A calculation derived from the slope of the UVB spectra reveals that a 1-nm shift of the solar UV spectrum to shorter wavelengths would result in a 35% increase in its mutagenic potential. At 325 nm, both biological action spectra show sharp decreases in slope. In addition, above 325 nm the spectra for lethality. mutagenicity, and dimer formation diverge sharply; lethalities at these UVA wavelengths were approximately tenfold greater relative to mutagenicity than at shorter wavelengths. The relative yield of dimer formation by 365 nm radiation is intermediate between the yields for lethality and mutagenesis.     

MULTIPLICATIVE ACTION OF A UV-B PHOTORECEPTOR and PHYTOCHROME IN ANTHOCYANIN SYNTHESIS

Using 290-nm light, which excites only a UV-B photoreceptor, and 385- and 660-nm light, which activate only phytochrome, the fluence rate-response curves of monochromatic irradiations for anthocyanin synthesis in the first internodes of broom sorghum (Sorghum bicolor Moench, cv. Acme Broomcorn) were analyzed. Although the two photoreceptors absorbed light independently, they multiplicatively increased the action of each other. Accordingly, when the fluence rates of both wavelengths were changed together, the resulting slopes of the fluence rate-response curves of double-log plots were steep compared with the slopes obtained with the respective monochromatic irradiations. The slopes of fluence rate-response curves for monochromatic irradiations at 325 to 345 nm were steeper than those at other wavelengths. This difference was shown to be due to the multiplicative actions of both photoreceptors.     

PHOTOSENSITIZATION WITH A CHLORIN-THIOBARBITURATE CONJUGATE

Abstract— The photobiologic properties of a chlorin-thiobarbiturate conjugate were examined. In addition to the characteristic 670 nm chlorin absorbance, the conjugate exhibited new absorbance bands at 517 and 743 nm, but these were not a part of the fluorescence emission spectra, nor was fluorescence observed at wavelengths >670 nm. The action spectrum indicated that the long-wavelength absorbance of the conjugate was not involved in phototoxicity in cell culture, and that only irradiation at wavelengths associated with the chlorin moiety yielded a cytotoxic effect. In vivo cytotoxicity elicited by 745 nm irradiation is attributed to thermal effects.     

Outdoor ultraviolet polychromatic action spectra for growth responses of Bellis perennis and Cynosurus cristatus

Polychromatic ultraviolet (UV) action spectra for various growth responses of the dicotyledon Bellis perennis L. (daisy) and the grass Cynosurus cristatus L. (crested dog's-tail) have been measured. The plants were grown in the natural environment and ambient daylight was supplemented with five different UV irradiances centred at eight different wavelengths (313, 318, 320, 322, 339, 348, 356 and 377 nm). Destructive growth analysis was performed on B. perennis and C. cristatus after 300 and 122 days respectively. Dose response curves were created to construct action spectra for individual responses. Different spectral responses were observed in these two plant types. B. perennis exhibited a substantial action maximum at 313 nm for the inhibition of aerial, root and total dry weight; a similar action maximum at 313 nm for the inhibition of leaf expansion was observed. Longer wavelengths were relatively ineffective on these growth parameters, with the exception of a small but statistically significant (P < 0.05) response to 320 nm radiation. By contrast, C. cristatus showed negligible response to 313 nm radiation, for inhibition of aerial, root and total dry weight but substantial responses to longer wavelengths, especially at 339 and 348 nm. These action spectra add weight to suggestions in the literature that UV-A has a role to play in responses in this region of the spectrum. The possible implications of these observations are discussed.     

Similarity in dose responses, action spectra and red light responses between phototropism and photoinhibition of growth.

Abstract— First positive phototropism and photoinhibition of growth of oat colcoptiles share similar dose response curves and action spectra. Both responses increase with increasing dosage of blue light (440 nm) up to 10¹³ photons cm^-2, then both decrease with increasing dosage. Action spectra for both responses have peaks at 360, 440, and 470 nm. When red light (660 nm) was given beforehand, the sensitivity of each response to blue light was lessened. These data indicate a close correlation between phototropism and photoinhibition of growth. Both phenomena can be explained as a result of photoinhibition of basipetal transport of auxin.