Light-dependent Proton Excretion of Wheat (Triticum aestivum L.) and Maize (Zea mays L.) Roots

We investigated the change of root net proton excretion of seedlings of Triticum aestivum L. and Zea mays L. with daily variation of illumination using a multi-channel pH-stat system. We found an increase of net proton excretion during darkness and a drop after the beginning of illumination. Inhibition of carotenoid biosynthesis by norflurazone and photooxidation of chlorophylls did not change the periodicity or its induction. The induction of diurnal periodicity was possible with blue, green and red light. After induction the oscillation of net proton excretion continued for at least two cycles under constant light. We conclude that net H⁺ excretion of wheat and maize roots may be regulated by an endogenous clock or by a signal from the leaves. The nature of such a hypothetical signal remains unknown.     

Connectivity of the retinal Schiff base to Asp85 and Asp96 during the bacteriorhodopsin photocycle: the local-access model.

In the recently proposed local-access model for proton transfers in the bacteriorhodopsin transport cycle (Brown et al. 1998. Biochemistry. 37:3982-3993), connection between the retinal Schiff base and Asp85 (in the extracellular direction) and Asp96 (in the cytoplasmic direction)is maintained as long as the retinal is in its photoisomerized state. The directionality of the proton translocation is determined by influences in the protein that make Asp85 a proton acceptor and, subsequently, Asp96 a proton donor. The idea of concurrent local access of the Schiff base in the two directions is now put to a test in the photocycle of the D115N/D96N mutant. The kinetics had suggested that there is a single sequence of intermediates, L<-->M1<-->M2<-->N, and the M2-->M1 reaction depends on whether a proton is released to the extracellular surface. This is now confirmed. We find that at pH 5, where proton release does not occur, but not at higher pH, the photostationary state created by illumination with yellow light contains not only the M1 and M2 states, but also the L and the N intermediates. Because the L and M1 states decay rapidly, they can be present only if they are in equilibrium with later intermediates of the photocycle. Perturbation of this mixture with a blue flash caused depletion of the M intermediate, followed by its partial recovery at the expense of the L state. The change in the amplitude of the C=O stretch band at 1759 cm-1 demonstrated protonation of Asp85 in this process. Thus, during the reequilibration the Schiff base lost its proton to Asp85. Because the N state, also present in the mixture, arises by protonation of the Schiff base from the cytoplasmic surface, these results fulfill the expectation that under the conditions tested the extracellular access of the Schiff base would not be lost at the time when there is access in the cytoplasmic direction. Instead, the connectivity of the Schiff base flickers rapidly (with the time constant of the M1<-->M2 equilibration) between the two directions during the entire L-to-N segment of the photocycle.     

The prolonged depolarizing afterpotential and its contribution to the understanding of photoreceptor function

Comparison of flies bred on vitamine A-poor and vitamine A-rich diets show the latter to exhibit, after blue illumination, 1) slight deviation from the linear relationship between stimulus intensity and receptor sensitivity and, 2) after intense blue illumination the phenomenon of the PDA. Both these effects could result from reduced pigment distances in such membranes. Maximum PDA was produced after about 20 s of illumination with blue light, and following this the resistance of the membrane was seen to stay low, returning to the resting value at the same rate as the PDA decline. The response to test flashes, repressed during illumination, gradually returned during the decline of the PDA, similar to the way the photoreceptor would respond to the sum of two stimuli: the test flash and a decreasing background illumination. Red light immediately following blue abolished the PDA and white light produced a small PDA. All these experiments corroborate a new model (without resorting to the concept of inhibitors) which links the photopigments with receptor excitation, the assumptions for which are the following: 1) PDA is produced after abnormally high primary quantum absorption by rhodopsin molecules, 2) PDA is a retarded membrane excitation by a substance in stored form, 3) the store is built up when production of this substance is larger than its consumption, and 4) time and energy are necessary for the regeneration of excitatory rhodopsin molecules.This work was supported by the DFG (Ha 258/10) and by the SFB 114Presented at the EMBO-Workshop on Transduction Mechanism of Photoreceptors, Jülich, Germany, October 4–8, 1976     

Proteorhodopsin is a light-driven proton pump with variable vectoriality

Proteorhodopsin, a homologue of archaeal bacteriorhodopsin (BR), belongs to a newly identified family of retinal proteins from marine bacteria, which could play an important role in the energy balance of the biosphere. We cloned the cDNA sequence of proteorhodopsin by chemical gene synthesis, expressed the protein in Escherichia coli cells, purified and reconstituted the protein in its functional active state. The photocycle characteristics were determined by time-resolved absorption and Fourier transform infrared (FT-IR) spectroscopy. The pH-dependence of the absorption spectrum indicates that the pK(a) of the primary acceptor of the Schiff base proton (Asp97) is 7.68. Generally, the photocycle of proteorhodopsin is similar to that of BR, although an L-like photocycle intermediate was not detectable. Whereas at pH>7 an M-like intermediate is formed upon illumination, at pH 5 no M-like intermediate could be detected. As the photocycle kinetics do not change between the acidic and alkaline state of proteorhodopsin, the only difference between these two forms is the protonation status of Asp97. This is corroborated by time-resolved FT-IR spectroscopy, which demonstrates that proton transfer from the retinal Schiff base to Asp97 is observed at alkaline pH, but the other vibrational changes are essentially pH-independent.After reconstitution into proteoliposomes, light-induced proton currents of proteorhodopsin were measured in a compound membrane system where proteoliposomes were adsorbed to planar lipid bilayers. Our results show that proteorhodopsin is a light-driven proton pump with characteristics similar to those of BR at alkaline pH. However, at acidic pH, the direction of proton pumping is inverted. Complementary experiments were carried out on proteorhodopsin expressed heterologously in Xenopus laevis oocytes under voltage clamp conditions.The following results were obtained. (1) At alkaline pH, proteorhodopsin mediates outwardly directed proton pumping like BR. (2) The direction of proton pumping can be inverted, when Asp97 is protonated. (3) The current can be inverted by changes of the polarity of the applied voltage. (4) The light intensity-dependence of the photocurrents leads to the conclusion that the alkaline form of proteorhodopsin shows efficient proton pumping after sequential excitation by two photons.     

Effects of Mutations of Lys41 and Asp102 of Bacteriorhodopsin

Bacteriorhodopsin (BR) is a retinal protein that functions as a light-driven proton pump. In this study, six novel mutants including K41E and D102K, were obtained to verify or rule out the possibility that residues Lys41 and Asp102 are determinants of the time order of proton release and uptake, because we found that the order was reversed in another retinal protein archaerhodopsin 4 (AR4), which had different 41th and 102th residues. Our results rule out that possibility and confirm that the pK _a of the proton release complex (PRC) determines the time order. Nevertheless, mutations, especially D102K, were found to affect the kinetics of proton uptake substantially and the pK _a of Asp96. Compared to the wild-type BR (BR-WT), the decay of the M intermediate and proton uptake in the photocycle was slowed about 3-fold in D102K. Hence those residues might be involved in proton uptake and delivery to the internal proton donor.     

Blue light effects on stomata are mediated by the guard cell plasma membrane redox system distinct from the proton translocating ATPase

Abstract. The effects of blue light on stomata are critically analysed. Blue-light-induced increase in stomatal conductance is preceded by membrane hyperpolarization, proton efflux, potassium uptake and malate synthesis in guard cells. Hypothetically, a flavin containing plasma membrane redox system can pump protons out of guard cells on illumination with blue light. It is proposed that this electrogenic proton pump requires NAD(P)H but does not involve ATP/ATPase.     

Kinetics of Nicotinamide Adenine Dinucleotides during Dark-Light Transients in Chlorella

Nicotinamide adenine dinucleotides were extracted and assayed at frequent intervals for 30 seconds prior to and 60 seconds immediately following illumination of Chlorella cells. For these experiments a special illumination vessel was designed which dispensed 1 ml aliquots of algae with no dead volume. —White incandescent illumination of aerated Chlorella caused an immediate oxidation of its pyridine nucleotides. Under similar conditions blue light caused an initial reduction of these coenzymes. — White illumination of the alga perfused with CO₂-free air caused a transitory reduction of the pyridine nucleotide followed by an oxidation and a second reduction, Blue light, under similar conditions caused an immediate reduction but no oxidation as seen with white light. — When suspensions were perfused with N₂, illumination produced a prompt and rapid oxidation of NADH. The extent of this oxidation was greater in white than in blue light. Under these conditions NADP⁺ was initially reduced and subsequently, temporarily oxidised. — The evidence offers some support for Lundegårdh's theory that blue light is more effective than red light in the photo-reduction of NADP⁺. The results are discussed with referemce to the demonstrated in vitro reduction of nicotinamide adenine dinucleotides by chloroplasts and the conflicting reports from in vivo kinetic studies of living photosynthetic organisms. A scheme is proposed to explain the nicotinamide adenine dinucleotide kinetics as observed in these experiments.     

Mono‐ and dichromatic LED illumination leads to enhanced growth and energy conversion for high‐efficiency cultivation of microalgae for application in space

Illumination with red and blue photons is known to be efficient for cultivation of higher plants. For microalgae cultivation, illumination with specific wavelengths rather than full spectrum illumination can be an alternative where there is a lack of knowledge about achievable biomass yields. This study deals with the usage of color LED illumination to cultivate microalgae integrated into closed life support systems for outer space. The goal is to quantify biomass yields using color illumination (red, blue, green and mixtures) compared to white light. Chlamydomonas reinhardtii was cultivated in plate reactors with color compared to white illumination regarding PCE, specific pigment concentration and cell size. Highest PCE values were achieved under low PFDs with a red/blue illumination (680 nm/447 nm) at a 90 to 10% molar ratio. At higher PFDs saturation effects can be observed resulting from light absorption characteristics and the linear part of PI curve. Cell size and aggregation are also influenced by the applied light color. Red/blue color illumination is a promising option applicable for microalgae‐based modules of life support systems under low to saturating light intensities and double‐sided illumination. Results of higher PCE with addition of blue photons to red light indicate an influence of sensory pigments.     

THE INVOLVEMENT OF A PLASMA MEMBRANE H+‐ATPase IN THE BLUE‐LIGHT ENHANCEMENT OF PHOTOSYNTHESIS IN LAMINARIA DIGITATA (PHAEOPHYTA)1

Many brown algae, including the kelp Laminaria digitata (Huds.) Lamour., exhibit enhanced photosynthesis when they are given a small amount of blue‐light in addition to a background of saturating red light. This blue light effect is correlated with an increased uptake of carbon. In the present study, we tested the hypothesis that blue light acts by increasing the activity of a plasma membrane H ⁺ ‐ATPase, thereby promoting an active carbon uptake across the plasma membrane. Photosynthetic carbon uptake was studied in pH‐drift experiments under illumination with red and blue light and using different inhibitors. Vanadate, an inhibitor of plasma membrane H ⁺ ‐ATPases, had a minor inhibitory effect on carbon uptake rates under saturating red light conditions, but inhibited the blue‐light enhancement by approximately 60%. An inhibitor of external carbonic anhydrase, acetazolamide, decreased the carbon uptake in both red light and in red plus blue light by 48% and 68%, respectively. These results suggest that photosynthetic carbon uptake depends on an external carbonic anhydrase under both red and red plus blue light conditions, and that blue light induces an increased activity of a P‐type H ⁺ ‐ATPase in the plasma membrane. The proton buffer Tris, which has a buffering capacity similar to vanadate in seawater, had no inhibitory effect on carbon uptake rates neither in red light nor in red plus blue light, showing that the inhibitory effect of vanadate is not caused by its effect as a buffer. The blue‐light enhancement was also abolished by a protein kinase inhibitor (H‐7), suggesting that the transduction of the blue‐light signal involves a protein kinase, which activates the plasma membrane H ⁺ ‐ATPase by phosphorylation.     

Crystal structures of acid blue and alkaline purple forms of bacteriorhodopsin

Bacteriorhodopsin, a light-driven proton pump found in the purple membrane of Halobacterium salinarum, exhibits purple at neutral pH but its color is sensitive to pH. Here, structures are reported for an acid blue form and an alkaline purple form of wild-type bacteriorhodopsin. When the P622 crystal prepared at pH 5.2 was acidified with sulfuric acid, its color turned to blue with a pKa of 3.5 and a Hill coefficient of 2. Diffraction data at pH 2-5 indicated that the purple-to-blue transition accompanies a large structural change in the proton release channel; i.e. the extracellular half of helix C moves towards helix G, narrowing the proton release channel and expelling a water molecule from a micro-cavity in the vicinity of the retinal Schiff base. In this respect, the acid-induced structural change resembles the structural change observed upon formation of the M intermediate. But, the acid blue form contains a sulfate ion in a site(s) near Arg82 that is created by re-orientations of the carboxyl groups of Glu194 and Glu204, residues comprising the proton release complex. This result suggests that proton uptake by the proton release complex evokes the anion binding, which in turn induces protonation of Asp85, a key residue regulating the absorption spectrum of the chromophore. Interestingly, a pronounced structural change in the proton release complex was also observed at high pH; i.e. re-orientation of Glu194 towards Tyr83 was found to take place at around pH 10. This alkaline transition is suggested to be accompanied by proton release from the proton release complex and responsible for rapid formation of the M intermediate at high pH.     

Different stomatal responses of maize leaves after blue or red illumination under anoxia

Abstract In normal air, illumination with a low level of blue or red light (40 μmol m^?2 s^?1) did not induce stomatal opening in maize plantlets. In CO₂-free air, 40 μmol m^?2 s^?1 of blue or red light promoted an enhancement in stomatal opening. At the same quantum flux, blue light was more efficient than red light and stomatal closure occurred more rapidly with a significantly shorter lag phase after blue light. Anoxia inhibited light-dependent stomatal opening, even under 320 μmol m^?2 s^?1 illumination. However, after 60 min of illumination with 40 μmol m^?2 s^?1 of blue light in anoxia, transient stomatal opening was observed when the plant was returned to darkness and normal air. This transient stomatal opening was weaker after pretreatment with red light. We conclude that a blue-light-dependent process induced under anoxia leads to stomatal opening provided oxygen is present. Possible mechanisms associated with blue-light-effect and the nature of the oxygen-consuming processes are discussed.     

Redox Processes in the Blue Light Response of Guard Cell Protoplasts of Commelina communis L

Guard cell protoplasts from Commelina communis L. illuminated with red light responded to a blue light pulse by an H⁺ extrusion which lasted for about 10 minutes. This proton extrusion was accompanied by an O₂ uptake with a 4H⁺ to O₂ ratio. The response to blue light was nil in darkness without a preillumination period of red light and increased with the duration of the red light illumination until about 40 minutes. However, acidification in response to a pulse of blue light was obtained in darkness when external NADH (1 millimolar) was added to the incubation medium, suggesting that redox equivalents necessary for the expression of the response to blue light in darkness may be supplied via red light. In accordance with this hypothesis, the photosystem II inhibitor 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (10 micromolar) decreased the acidification in response to blue light more efficiently when it was added before red light illumination than before the blue light pulse. In the presence of hexacyanoferrate, the acidification in response to a blue light pulse was partly inhibited (53% of control), suggesting a competition for reducing power between ferricyanide reduction and the response to blue light.     

Polarographische Messung der Nitritreduktion von Chlorella im monochromatischen Licht

Summary In green plant cells nitrite is reduced by two systems, one dependent on photosynthesis and the other upon respiration. Using a polarographic method for continuous measurement of nitrite uptake, the relationship between light driven and respiration linked nitrite reduction of Chlorella cells was studied.Photosynthetic nitrite reduction is characterized by a pronounced increase in the velocity of nitrite uptake upon illumination. After the light is turned off the velocity immediately returns to the preillumination value. Photosynthetic nitrite reduction of Chlorella is separated from respiration linked nitrite reduction by illumination with red light under anaerobic conditions; it is stimulated by CO₂ and is inhibited by DCMU, findings which confirm earlier observations.In white light a special blue light stimulation of nitrite uptake is overlapped by photosynthetic nitrite reduction. In contrast to photosynthetic nitrite reduction this type of light stimulation is characterized by a lag period of about I min from the onset of illumination; it continues about 10 min when the light is turned off. It is separated from photosynthetic nitrite reduction by irradiation of the algae with low intensities of short wavelength light (<500 nm). Blue light stimulation of nitrite uptake of Chlorella is strongly dependent on the developmental stage of the cells. It is observed with young cells (autospores) of synchronized algae only.There is no evidence for any connection between blue light stimulation of nitrite uptake and photosynthesis. From the sensitivity of this process towards anaerobic conditions and antimycin A it is concluded to be a stimulation of respiration linked nitrite reduction.Under conditions of low exogenous nitrite concentration a temporary inhibition of steady state dark nitrite reduction appears immediately after the light is turned off. From several observations it is concluded that the inhibition already exists during the preceding illumination and decreases the rate of total nitrite uptake in the light. This process is suppressed by inhibition of respiration as well as by the inhibitor of photosynthesis, DCMU.If nitrate is the source of nitrogen an excretion of nitrite is found following illumination. The kinetics of this process agree with those observed for the light induced inhibition of steady state dark nitrite reduction immediately after illumination.     

Blue light- and genetically-reversed gravitropic response in protonemata of the moss Ceratodon purpureus

In darkness, protonemal filaments of Ceratodon purpureus (Brid.) grow negatively gravitropically (upwards). Red light induces a positive phototropic response mediated by the photoreceptor phytochrome. A red light treatment also has an inhibitory effect on the gravitropic response, an effect also mediated by phytochrome. In this study the effects of blue light on phototropism and on gravitropism were analysed. Unilateral blue light resulted in only a weak phototropic response, but markedly randomised growth direction. Blue light given together with a gravitropic stimulus reversed the gravitropism, changing it from negative to positive (filaments grow downward). The effect of blue light was also analysed with the mutant ptr116, which is defective in the biosynthesis of the phytochrome chromophore, and in a newly isolated mutant wwr2, which is positively gravitropic in darkness. Blue light induced the same reversal of gravitropism in ptr116 as in the wild type, indicating that phytochrome is not involved in this process. In wwr2 the direction of gravitropism was unaltered by the blue light treatment. Light also affects chlorophyll content and the size of plastids, potential statoliths for gravitropism. Red light induced an increase in plastid size and chlorophyll content in the wild type but not in ptr116. Blue light induced a similar change in wild type plastids. It seems as though light-induced alterations of gravitropism are not simply mediated by alterations in plastid properties, and that red light and blue light evoke fundamentally different responses. Received: 11 July 1997 / Accepted: 30 January 1998     

Electrooptical studies on proton-binding and -release of bacteriorhodopsin

Electric field induced pH changes of purple membrane suspensions were investigated in the pH range from 4.1 to 7.6 by measuring the absorbance change of pH indicators. In connection with the photocycle and proton pump ability, three different states of bacteriorhodopsin were used: (1) the native purple bacteriorhodopsin (magnesium and calcium ions are bound, the M intermediate exists in the photocycle and protons are pumped), (2) the cation-depleted blue bacteriorhodopsin (no M intermediate), and (3) the regenerated purple bacteriorhodopsin which is produced either by raising the pH or by adding magnesium ions (the M intermediate exists). In the native purple bacteriorhodopsin there are, at least, two types of proton binding sites: one releases protons and the other takes up protons in the presence of the electric field. On the other hand, blue bacteriorhodopsin and the regenerated purple bacteriorhodopsin (pH increase) show neither proton release nor proton uptake. When magnesium ions are added to the suspensions; the field-induced pH change is observed again. Thus, the stability of proton binding depends strongly on the state of bacteriorhodopsin and differences in proton binding are likely to be related to differences in proton pump activity. Furthermore, it is suggested that the appearance of the M intermediate and proton pumping are not necessarily related.     

Crystal structure of the O intermediate of the Leu93→Ala mutant of bacteriorhodopsin

The lifetime of the O intermediate of bacteriorhodopsin (BR) is extended by a factor of ～250 in the Leu93‐to‐Ala mutant (BR_L93A). To clarify the structural changes occurring in the last stage of the proton pumping cycle of BR, we crystallized BR_L93A into a hexagonal P622 crystal. Diffraction data from the unphotolyzed state showed that the deletion of three carbon atoms from Leu93 is compensated by the insertion of four water molecules in the cytoplasmic vicinity of retinal. This insertion of water is suggested to be responsible for the blue‐shifted λ_max (540 nm) of the mutant. A long‐lived substate of O with a red‐shifted λ_max (～565 nm) was trapped when the crystal of BR_L93A was flash‐cooled after illumination with green light. This substate (O_slow) bears considerable similarity to the M intermediate of native BR; that is, it commonly shows deformation of helix C and the FG loop, downward orientation of the side chain of Arg82, and disruption of the Glu194/Glu204 pair. In O_slow, however, the main chain of Lys216 is less distorted and retinal takes on the 13‐cis/15‐syn configuration. Another significant difference is seen in the pH dependence of the structure of the proton release group, the pK_a value of which is suggested to be much lower in O_slow than in M. Proteins 2012;. © 2012 Wiley Periodicals, Inc.     

Mechanism of proton transport in bacteriorhodopsin from crystallographic structures of the K,L, M1, M2, and M2' intermediates of the photocycle

We produced the L intermediate of the photocycle in a bacteriorhodopsin crystal in photo-stationary state at 170 K with red laser illumination at 60% occupancy, and determined its structure to 1.62 A resolution. With this model, high-resolution structural information is available for the initial bacteriorhodopsin, as well as the first five states in the transport cycle. These states involve photo-isomerization of the retinal and its initial configurational changes, deprotonation of the retinal Schiff base and the coupled release of a proton to the extracellular membrane surface, and the switch event that allows reprotonation of the Schiff base from the cytoplasmic side. The six structural models describe the transformations of the retinal and its interaction with water 402, Asp85, and Asp212 in atomic detail, as well as the displacements of functional residues farther from the Schiff base. The changes provide rationales for how relaxation of the distorted retinal causes movements of water and protein atoms that result in vectorial proton transfers to and from the Schiff base.     

Photoinactivation of Acinetobacter baumannii and Escherichia coli B by a Cationic Hydrophilic Porphyrin at Various Light Wavelengths

Photodynamic treatment by the cationic TMPyP photosensitizer was undertaken on the multiple antibiotic-resistant bacteria Acinetobacter baumannii and Escherichia coli. Total eradication of the bacterial cultures was determined immediately after initiation of illumination when these bacteria were treated with 5, 10, 15, 20-tetra (4-N methylpyridyl)porphine (TMPyP) at a concentration of 29.4 μmol/L and illuminated by blue, green, or red light. Total eradication of both bacteria was obtained also after treatment of bacterial cultures with 3.7 μmol/L TMPyP and illumination with blue light (400–450 nm). On the other hand, an 8- or 16- to 20-fold higher light intensity, respectively, was required for total eradication upon illumination with green (480–550 nm) or red light (600–700 nm). A 407-nm blue light only 7 and 9 joules/cm², respectively, was needed for total eradication of both bacteria even at a concentration of 3.7 μmol/L TMPyP. X-ray-linked microanalysis demonstrated loss of potassium and a flood of sodium and chloride into the cells, indicating serious damage to the cytoplasmic membrane. Transmission electron microscopy (TEM) revealed structural changes and damage to the membrane of treated E. coli. In A. baumannii-treated cells, mesosomes and black dots that resemble aggregation of polyphosphate polymers could be seen. DNA breakage appeared only after a long period of illumination, when the bacterial cell was no longer viable. It can be concluded that cytoplasmic membrane damage and not DNA breakage is the major cause for bacterial death upon photosensitization. Received: 13 October 2000 / Accepted: 17 November 2000     

The biphasic fluence response of carotenogenesis in Neurospora crassa: Temporary insensitivity of the photoreceptor system

Whether or not illumination is continuous or interrupted during the span in which increasing illumination time periods (i.e., increasing fluences) have no different effect on carotenogenesis is optional, in regard to the amount of carotenoids produced (revealing the plateau of the biphasic fluence response curve). This indicates temporary insensitivity. When the time delay between the onsets of the initial and second illumination is extended beyond the expanse of the plateau, the amount of carotenoids induced by the second illumination depends on the time elapsed following the first exposure; after ca. 2 h, maximum competence for a second induction is completely restored. On the other hand, the amount of carotenoids induced by a second illumination also depends on the duration of this second illumination, but, unlike the dependence in a single illumination, results in a different fluence response curve for the second exposure. When UV-A is used for induction, the refractory period which follows the first exposure seems to be the same as for blue light, suggesting vision of UV-A and blue light by the same photoreceptor system.