DARK ADAPTATION AND THE DARK GROWTH RESPONSE OF PHYCOMYCES

Light-adapted sporangiophores of the fungus Phycomyces respond to sudden darkening by a temporary decrease in the rate of elongation, after a latent period of several minutes. The reaction time of this "dark growth" response is compound like that of the "light growth" response. It is, moreover, shorter the more intense the previous illumination. The rate of dark adaptation following adaptation to a very large range of light intensities is found to be proportional to the logarithm of the preceding light intensity. It is shown that a constant amount of dark adaptation takes place before the response occurs. On the assumption that changes in the rate of growth reflect changes in the concentration of a substance which at constant light intensity is in equilibrium with a light-sensitive material, possible equations for such a photostationary state are examined. The most reasonable formulation requires that the partial velocity of the "light" reaction be taken proportional to log I instead of to I directly.     

Photosensory Adaptation in Plants

Photosensory adaptation (range adjustment) is of great importance in plants (including fungi and various microorganisms) that operate in large intensity ranges. The adaptation mechanisms of plants have some features in common with those of vertebrates and invertebrates. As with those systems, plants have biphasic exponential dark-adaptation kinetics, that are much slower than the corresponding light adaptation kinetics. One needs to distinguish between sensor adaptation, which regulates range adjustment, and effector adaptation (habituation), which regulates the motor apparatus of the organism (flagellar movement or cell wall growth). In Phycomyces, and perhaps Stentor, sensor adaptation is mediated by the photoreceptor system. In contrast to vertebrates and invertebrates, dark adaptation can be controlled in some plants by special photoreceptors. In Phycomyces, these can be either photo-products of the actinic photoreceptor(s) or not yet identified receptor pigments. In higher plants phytochrome can alter the state of adaptation.     

DARK ADAPTATION AND THE LIGHT-GROWTH RESPONSE OF PHYCOMYCES

1. A single-celled, elongating sporangiophore of Phycomyces responds to a sufficient increase in intensity of illumination by a brief increase in growth rate. This is the "light-growth response" of Blaauw. 2. The reaction time is compound, consisting of an exposure period and a latent period (this comprising both the true latent period resulting from photochemical action and any "action time" necessary for the response). During the latter period the plant may be in darkness, responding nevertheless at the end of the latent period. 3. Both light adaptation and dark adaptation occur in the sporangiophore. The kinetics of dark adaptation can be accounted for on the basis of a bimolecular reaction, perhaps modified by autocatalysis. Attention is called to the bimolecular nature of the "dark" reaction in all other photosensory systems that have been studied, in spite of the diversity of the photosensitive substances themselves and of the different forms of the responses to light.     

A Kinetic Model for Adaptation and the Light Responses of Phycomyces

A kinetic model is described consisting of two sequential first order processes connected by two parallel reaction pathways, one of which is light-catalyzed. A change in light flux changes the rate constant of the light-dependent process, whereupon the levels of two chemical intermediaries readjust. The model's output duplicates all the main features of the cell's light-growth and dark-growth responses except their latent periods. An asymmetric modification of the model reproduces the two types of phototropic inversion discovered by Reichardt and Varjú and by Dennison. Simple exponential equations describe these responses of the model, as well as the theoretical course of its light and dark adaptation. It is concluded that adaptation in Phycomyces consist in the photocatalytic adjustment of the level of a metabolic reservoir.     

Presence of GTP-Binding Proteins in the Plasma Membrane of the Phycomyces Sporangiophore

Ashktorab, H., and Cohen, R. J. 1994. Presence of GTP-binding proteins in the plasma membrane of the Phycomyces sporangiophore. Experimental Mycology 18, 139-149. When a plasma membrane-enriched fraction isolated from the sporangiophore of the Zygomycete Phycomyces blakesleeanus was subject to immunoblotting, two polypeptide bands reacted with an antibody directed to a conserved sequence of the ∝ subunit of G-proteins; their apparent molecular masses were 40 and 51 kDa. Upon treating the plasma membrane preparation with cholera toxin, bands at 40 and 51 kDa appeared to be ADP-ribosylated but no band appeared with pertussis toxin incubation. Apparent dissociation constants for the binding of GTPγS were determined for plasma membrane from Phycomyces sporangiophore grown in the light (K_D = 39 ± 16 nM) (±SD) and in the dark (K_D = 11 ± 6 nM). GTP served as a strong competitor for binding as did GDP, although somewhat less well, while ATP competed considerably more weakly. Northern analysis of sporangiophore mRNA displayed two bands hybridizing to the Gα2 probes coding for a G_α subunit from Dictyostelium discoideum. Furthermore, Western blotting of plasma membrane revealed several bands containing polypeptides with presumptive covalently attached immunoreactive flavins. (The prevailing evidence from the action spectra of Phycomyces is that the photoreceptor is a flavoprotein residing in the plasma membrane.) Immunoblotting also detected a H⁺ ATPase similar to the plasma membrane enzyme of yeast, corroborating our isolation of plasma membrane and suggesting another possible player in the signal responses of Phycomyces . This is apparently the first evidence for a G-protein in this class of eukaryotes. G-proteins may serve a role in the flavoprotein-mediated phototransduction system of P. blakesleeanus.     

Adaptation in the Ventral Eye of Limulus is Functionally Independent of the Photochemical Cycle, Membrane Potential, and Membrane Resistance

The early receptor potential (ERP), membrane potential, membrane resistance, and sensitivity were measured during light and/or dark adaptation in the ventral eye of Limulus. After a bright flash, the ERP amplitude recovered with a time constant of 100 ms, whereas the sensitivity recovered with an initial time constant of 20 s. When a strong adapting light was turned off, the recovery of membrane potential and of membrane resistance had time-courses similar to each other, and both recovered more rapidly than the sensitivity. The receptor depolarization was compared during dark adaptation after strong illumination and during light adaptation with weaker illumination; at equal sensitivities the cell was more depolarized during light adaptation than during dark adaptation. Finally, the waveforms of responses to flashes were compared during dark adaptation after strong illumination and during light adaptation with weaker illumination. At equal sensitivities (equal amplitude responses for identical flashes), the responses during light adaptation had faster time-courses than the responses during dark adaptation. Thus neither the photochemical cycle nor the membrane potential nor the membrane resistance is related to sensitivity changes during dark adaptation in the photoreceptors of the ventral eye. By elimination, these results imply that there are (unknown) intermediate process(es) responsible for adaptation interposed between the photochemical cycle and the electrical properties of the photoreceptor.     

The Duplex Nature of the Retina of the Nocturnal Gecko as Reflected in the Electroretinogram

The effect of light and dark adaptation on the electrical activity in two species of nocturnal gecko, Hemidactylus turcicus and Tarentola mauritanica was studied. The electroretinogram of both species changes from the scotopic type in the dark-adapted state to the photopic type after strong light adaptation. For the scotopic response fusion frequencies up to 18 flashes per sec. are obtained in both species. For the photopic response fusion frequencies up to 50 flashes per sec. are seen in Tarentola, and up to 25 flashes per sec. in Hemidactylus. Proceeding from dark to light adaptation the increment threshold (dI) is measured at different levels of adaptive illumination (I). At low levels of illumination the dI/I ratio is found to be small and at high levels of illumination to be large. No difference in the dI/I ratio is obtained for test lights of 462 and 605 mµ. During dark adaptation the change of threshold after exposure to moderate and weak lights (up to 10³ times dark threshold) is rather fast. After light adaptation to strong light (10⁶ times dark threshold) duplex dark adaptation curves are seen with a break separating a fast and a slow phase of dark adaptation. The significance of these results from a retina which possesses sense cells of only one type is discussed.     

DARK ADAPTATION IN DINEUTES

The level of dark adaptation of the whirligig beetle can be measured in terms of the threshold intensity calling forth a response. The course of dark adaptation was determined at levels of light adaptation of 6.5, 91.6, and 6100 foot-candles. All data can be fitted by the same curve. This indicates that dark adaptation follows parts of the same course irrespective of the level of light adaptation. The intensity of the adapting light determines the level at which dark adaptation will begin. The relation between log aI ₀ (instantaneous threshold) and log of adapting light intensity is linear over the range studied.     

Changes of the chlorophyll fluorescence induction kinetics of C3 and CAM plants during day/night cycles

The induction kinetics of the 680 nm chlorophyll fluorescence were measured on attached leaves of Kalanchoë daigremontiana R. Hamet et Perr. (CAM plant), Sedum telephium L. and Sedum spectabile Bor. (C₃ plant in spring, CAM plant in summer) and Raphanus sativus L. (C₃ plant) at three different times during a 12/12h day/night cycle. During the fluorescence transient the fluorescence intensity at the O, P and T-level (f_O, f_max, f_st,) was different for the plant species tested; this may be due to their different leaf structure, pigment composition and organization of their photosystems. The kinetics of the fluorescence induction depended on the time of preillumination or dark adaptation during the light/dark cycle but not on the type of primary CO₂ fixation mechanism (C₃ and CAM). For dark adapted leaves measured either at the end of the dark phase or after dark adaptation of plants taken from the light phase a higher P-level fluorescence, a higher variable fluorescence (P-O) and a larger complementary area were found than for leaves of plants taken directly from the light phase. This indicates the presence of largely oxidized photosystem 2 acceptor pools during darkness. During the light phase the fluorescence decline after the P-level was faster than during the dark phase; from this we conclude that the light adaptation of the photosynthetic apparatus (state 1→ state 2 transition, Δ pH) during the induction period proceeded faster in plants taken from the light phase than in plants taken from the dark phase.     

DARK ADAPTATION IN AGRIOLIMAX

A method is described which measures the excitation of Agriolimax by light, during the progress of light adaptation, by assuming that the orientating effect of continuous excitation is expressed as a directly proportionate tension difference in the orienting muscles of the two sides of the body. The tendency toward establishment of such a tension difference is caused to work against a similar geotropic effect at right angles to the phototropic one. This enables one to study the kinetics of light adaptation, and of dark adaptation as well. The situation in the receptors is adequately described by the paradigm See PDF for Equation similar to that derived by Hecht for the differential sensitivity of various forms, but with the difference that the "dark" reaction is not only "bimolecular" but also autocatalysed by the reaction product S. The progress of dark adaptation is reflected (1) in the recovery of the amplitude of the orientation and (2) in the rates of light adaptation at different levels of the recovery; each independently supports these assumptions, for which the necessary equations have been provided. These equations also account for the relative variabilities of the angles of orientation, and, more significantly, for the two quite different kinds of curves of dark adaptation which are obtained in slightly different types of tests.     

Sensitivity and adaptation in the Drosophila phototransduction and photoreceptor degeneration mutants trp and rdgB

We studied rdgB, a retinal degeneration mutant, and trp, a phototransduction mutant, separately and in combination in Drosophila. First we showed that trp did not block degeneration in white-eyed rdgB mutants. Thus, rdgB was useful in determining the defects which trp caused in the compound eye receptors R7 and R8; this is because rdgB selectively eliminates R1-6 photoreceptors which would, if present, dominate the compound eye responses. R7 and R8 both express the trptransient receptor potential phenotype in trp mutants. The trp mutation does not change receptor spectral sensitivities, nor does it alter the dark stability of R1-6's and R7's metarhodopsins as judged by dark adaptation studies. The dark adaptation is not significantly affected by trp. However, trp slows the dark adaptation of R8 considerably and seems to make the blue-induced inactivation of R1-6 less stable.     

Growth of the morphological,piloboloid mutants ofPhycomyces blakesleeanus

The sporangiophore of a growth zone-defective mutant, piloboloid (genotypepil), ofPhycomyces gradually ceases longitudinal elongation and radially expands until the growth zone becomes nearly spherical, shortly after the sporangiophore has reached the final developmental stage. The growth of 23pil mutants, obtained by mutagenesis withN-methyl-N′-nitro-N-nitrosoguanidine (NTG), 2-methoxy-6-chloro-9-[3-(ethyl-2-chloroethyl)aminopropylamino]acridine-2HCl (ICR-170), or 4-nitroquinoline-1-oxide (NQO), was studied under different conditions. Allpil mutants obtained were genetically homokaryotic. Expansion in the growth zone and degree of synchrony ofpil sporangiophore length were both greater at low temperature (10°C) and under white light (10 μW/cm²) than at high temperature (20, 25°C) and in the dark. Dwarf sporangiophores of about 1-mm length, which characteristically stop growing immediately after sporangium formation, did not expand radially. The mycelial morphology ofpil mutants was almost normal compared with that of the wild type, though growth was some-what slower in the former than the latter.     

The xanthophyll cycle and NPQ in diverse desert and aquatic green algae

It has long been suspected that photoprotective mechanisms in green algae are similar to those in seed plants. However, exceptions have recently surfaced among aquatic and marine green algae in several taxonomic classes. Green algae are highly diverse genetically, falling into 13 named classes, and they are diverse ecologically, with many lineages including members from freshwater, marine, and terrestrial habitats. Genetically similar species living in dramatically different environments are potentially a rich source of information about variations in photoprotective function. Using aquatic and desert-derived species from three classes of green algae, we examined the induction of photoprotection under high light, exploring the relationship between nonphotochemical quenching and the xanthophyll cycle. In liquid culture, behavior of aquatic Entransia fimbriata (Klebsormidiophyceae) generally matched patterns observed in seed plants. Nonphotochemical quenching was lowest after overnight dark adaptation, increased with light intensity, and the extent of nonphotochemical quenching correlated with the extent of deepoxidation of xanthophyll cycle pigments. In contrast, overnight dark adaptation did not minimize nonphotochemical quenching in the other species studied: desert Klebsormidium sp. (Klebsormidiophyceae), desert and aquatic Cylindrocystis sp. (Zygnematophyceae), and desert Stichococcus sp. (Trebouxiophyceae). Instead, exposure to low light reduced nonphotochemical quenching below dark-adapted levels. De-epoxidation of xanthophyll cycle pigments paralleled light-induced changes in nonphotochemical quenching for species within Klebsormidiophyceae and Trebouxiophyceae, but not Zygnematophyceae. Inhibition of violaxanthin–zeaxanthin conversion by dithiothreitol reduced high-light-associated nonphotochemical quenching in all species (Zygnematophyceae the least), indicating that zeaxanthin can contribute to photoprotection as in seed plants but to different extents depending on taxon or lineage.     

Light response inPhycomyces blakesleeanus: Evidence for roles of chitin biosynthesis and breakdown

Chitin synthetase activity, both basal and zymogenic, fromPhycomyces sporangiophores was stimulated by lightin vitro andin vivo. AmadB mutant did not display these activations, whereas in amadE mutant only chitin synthetase zymogen was increased by illuminationin vivo. Light also produced a transient alteration in cell wall structure at the apical region of the sporangiophore revealed by accessibility of chitin to binding by wheat germ agglutinin and by an increased limited breakage of chitin microfibrils. This last response was absent in bothmadB andmadE mutants. Accordingly, it is suggested that the light growth response in the sporangiophore fromPhycomyces is due to a transient softening of the cell wall at the growing region followed by an elongation due to the turgor pressure of the cell and an enhanced chitin biosynthesis by the apically localized chitin synthetase which restores normal strength to the cell wall. A hypothetical scheme to account for these results is presented.     

Photocontrol of Voltage-Gated Ion Channel Activity by Azobenzene Trimethylammonium Bromide in Neonatal Rat Cardiomyocytes

The ability of azobenzene trimethylammonium bromide (azoTAB) to sensitize cardiac tissue excitability to light was recently reported. The dark, thermally relaxed trans- isomer of azoTAB suppressed spontaneous activity and excitation propagation speed, whereas the cis- isomer had no detectable effect on the electrical properties of cardiomyocyte monolayers. As the membrane potential of cardiac cells is mainly controlled by activity of voltage-gated ion channels, this study examined whether the sensitization effect of azoTAB was exerted primarily via the modulation of voltage-gated ion channel activity. The effects of trans- and cis- isomers of azoTAB on voltage-dependent sodium (INav), calcium (ICav), and potassium (IKv) currents in isolated neonatal rat cardiomyocytes were investigated using the whole-cell patch-clamp technique. The experiments showed that azoTAB modulated ion currents, causing suppression of sodium (Na⁺) and calcium (Ca²⁺) currents and potentiation of net potassium (K⁺) currents. This finding confirms that azoTAB-effect on cardiac tissue excitability do indeed result from modulation of voltage-gated ion channels responsible for action potential.     

Far-red light-induced changes in intracellular potentials of spinach mesophyll cells: interaction with red light

In green plants, the large bioelectric changes that photosynthetically active light stimulates make it difficult to observe electrical potential changes related to phytochrome photoconversion. As a first step towards distinguishing between photosynthetic and phytochrome effects, we showed that red light enhances far-red stimulated intracellular potential changes in spinach (Spinacia oleracea) leaf mesophyll cells.

For a dark-adapted leaf, the response to far-red light increased during the first 10 to 30 exposures of 2.5 minutes, after which it was constant. The intracellular potential depolarized by an average of 0.3 millivolts during each 2.5-minute far-red light period, and returned to the resting value during each subsequent dark period. Continuous supplementary red light (at 1-5% of the fluence rate of the far-red light that stimulated the depolarizations) increased the response to far-red 2- to 3-fold. Supplementary red light did not amplify the response to alternating 702 nanometers light and dark periods. The Emerson enhancement effect thus does not seem to explain amplification of the response to 730 nanometers light by supplementary red light. This does not prove that photosynthetic pigments are not involved in some other way.

     

ON "REVERSAL" OF PHOTOTROPISM IN PHYCOMYCES

Alleged reversal of the phototropism of the sporangiophores of Phycomyces by high intensities of light does not occur if infra-red radiation is properly excluded. Phototropic "indifference" alone occurs at high intensities due to equal photic action on both sides of the sporangiophore. If heat radiation is not screened out, a gradual, negative thermotropic bending takes place.     

Cyclic variations of photosynthetic activity under nitrogen fixing conditions in Synchococcus RF-1

When nitrogen fixing cell cultures of Synechococcus RF-1 were subjected to an alternating lightdark regime (12 h:12 h), a cyclic decrease in the photosynthetic oxygen evolution potential was observed during the dark periods. This rhythm of net photosynthesis rate was maintained for at least two days after transition to continuous light. The decrease in net photosynthesis was accompanied by a stimulation of dark respiration. However, the magnitude of oxygen uptake was considerably smaller than the observed decrease in oxygen evolution. The photosynthetic activity of cells taken from the dark period was characterized by (i) a significantly lower quantum yield and (ii) a strong reduction in the light-saturated rate of photosynthesis. Growing the cultures on nitrate or under continuous light completely suppressed this rhythm. Protein synthesis was not necessary for the recovery of the light-saturated rate of photosynthesis during the light period. The cellular content of chlorophyll a and of phycobiliproteins did not vary between light and dark period, indicating that quantitative changes in the composition of the photosynthetic apparatus are not the basis for the observed oscillations. Regulatory modifications of the photosynthetic efficiency are proposed as an adaptation mechanism to adjust the intracellular oxygen concentration to the needs for nitrogenase activity.Abbreviation Chl chlorophyll     

Neural and Photochemical Mechanisms of Visual Adaptation in the Rat

The effects of light adaptation on the increment threshold, rhodopsin content, and dark adaptation have been studied in the rat eye over a wide range of intensities. The electroretinogram threshold was used as a measure of eye sensitivity. With adapting intensities greater than 1.5 log units above the absolute ERG threshold, the increment threshold rises linearly with increasing adapting intensity. With 5 minutes of light adaptation, the rhodopsin content of the eye is not measurably reduced until the adapting intensity is greater than 5 log units above the ERG threshold. Dark adaptation is rapid (i.e., completed in 5 to 10 minutes) until the eye is adapted to lights strong enough to bleach a measurable fraction of the rhodopsin. After brighter light adaptations, dark adaptation consists of two parts, an initial rapid phase followed by a slow component. The extent of slow adaptation depends on the fraction of rhodopsin bleached. If all the rhodopsin in the eye is bleached, the slow fall of threshold extends over 5 log units and takes 2 to 3 hours to complete. The fall of ERG threshold during the slow phase of adaptation occurs in parallel with the regeneration of rhodopsin. The slow component of dark adaptation is related to the bleaching and resynthesis of rhodopsin; the fast component of adaptation is considered to be neural adaptation.     

Linear Dichroism and Orientation of the Phycomyces Photopigment

The greater sensitivity of a cylindrical Phycomyces sporangiophore to blue light polarized transversely rather than longitudinally is a consequence of the dichroism and orientation of the receptor pigment. The abilities of wild type and several carotene mutants to distinguish between the two directions of polarization are the same. The E-vector angle for maximum response relative to the transverse direction is 42 ± 4° at 280 nm, 7° ± 3° at 456 nm, and 7° ± 8° at 486 nm. The in vivo attenuation of polarized light at these wavelengths is very small. The polarized light effect in Phycomyces cannot arise from reflections at the cell surface or from differential attenuations due to internal screening or scattering.