Multiple-flash activation of the water-photolysis system in wheat leaves as observed by delayed emission.

The chloroplasts from wheat leaves greened under intermittent illuminations (1 ms in duration) at long intervals (5 min) are capable of photoreducing DCIP (2,6-dichlorophenolindophenol) with diphenylcarbazide as an electron donor but are incapable of photoreducing DCIP with water as the donor. On exposure of such intermittently illuminated leaves to flashes spaced at intervals of less than 10s, the delayed light emission from the leaves was greatly enhanced in parallel with the generation of Hill activity. The mechanism of this photoactivation was studied by following the changes of the delayed emission from intermittently illuminated leaves exposed to short-interval flashes programmed in various ways. Analysis of the kinetic data indicated that the photoactivation involves three consecutive photoreactions with a rate-limiting dark reaction between them; P-light leads to A0-light leads to A1-dark leads to A2-light leads to A3 in which P is a precursor convertible to A0, the first intermediate with a longer lifetime of t 1/2 approximately 100s and A3 is the final activated compound or state converted by short-interval flashes from A0 through A1 and A2, two other intermediates with shorter lifetimes of t 1/2 approximately 0.4s and 5s, respectively.     

Multiple-flash activation of the water-photolysis system in wheat leaves as observed by delayed emission

The chloroplasts from wheat leaves greened under intermittent illuminations (1 ms in duration) at long intervals (5 min) are capable of photoreducing DCIP (2, 6-dichlorophenolindophenol) with diphenylcarbazide as an electron donor but are incapable of photoreducing DCIP with water as the donor. On exposure of such intermittently illuminated leaves to flashes spaced at intervals of less than 10 s, the delayed light emission from the leaves was greatly enhanced in parallel with the generation of Hill activity. The mechanism of this photoactivation was studied by following the changes of the delayed emission from intermittently illuminated leaves exposed to short-interval flashes programmed in various ways. Analysis of the kinetic data indicated that the photoactivation involves three consecutive photoreactions with a rate-limiting dark reaction between them; P-light → A₀-light → A₁-dark → A₂-light → A₃ in which P is a precursor convertible to A₀, the first intermediate with a longer lifetime of $\text{t}\text{1}\text{2}\text{≈ 100}\text{s}$ and A₃ is the final activated compound or state converted by short-interval flashes from A₀ through A₁ and A₂, two other intermediates with shorter lifetimes of $\text{t}\text{1}\text{2}\text{≈ 0.4}\text{s}$ and 5 s, respectively.     

Action Spectrum for Photoactivation of the Water-splitting System in Plastids of Intermittently Illuminated Wheat Leaves

The chloroplasts from wheat leaves developed under intermittent illumination (1 ms light + 12 min dark) were able to photoreduce DPIP with DPC as electron donor but unable to photoreduce DPIP with water as electron donor. On exposure of these leaves to continuous light, the Hill activity with water as electron donor was rapidly induced. The photoactivation was sensitive to the treatment with DCMU prior to exposure to continuous light. The action spectrum for the photoactivation showed a sharp band at 680 nm with a distinct shoulder at 650 nm, and was similar to the absorption spectrum of photosytem-2 particles. These data suggest that the electron transfer driven by photosystem 2 is essential for the activation of the water-splitting system in the chloroplasts of intermittently illuminated leaves.     

Short-term reactions of phytochrome in Mougeotia?

Summary Phytochrome controlled chloroplast movement in Mougeotia is induced by flashes of polarized red light. Two subsequent flashes, separated by a dark interval of a few seconds, are much more effective than two simultaneous flashes; a maximal cumulative effect is reached if the duration of the dark interval is 30 ms or longer. We propose two light reactions in series, separated by a very fast dark reaction. Preliminary evidence is given that the energy requirement for these light reactions is different. It is suggested that the two reactions are related in some way to free and bound phytochrome.Dedicated to Prof. Dr. E. Bünning on the occasion of his seventieth birthday.     

Effects of priming flash parameters and dark interval on red-induced afterpotential in Balanus photoreceptors

The sequence (a) priming flash, (b) dark interval, and (c) red light induces a long-lasting afterdepolarization (PDA) in Balanus photoreceptors. The inward flow of membrane current associated with the decay of PDA was independent of red test flashes, provided that PDA had plateaued at a particular intensity. The influence of wavelength and duration of the priming flash and their interaction with the dark interval were investigated. Increasing the duration of the priming flash produced a systematic increase in PDA duration. The dark interval plays a crucial role in PDA induction. The priming flash duration and the dark interval were reciprocally related, i.e, short flashes followed by long dark intervals induced as much PDA as long priming flashes followed by short dark intervals. The action spectrum for the priming flash was found to correspond to that of the primary photopigment (VP537).     

Flash reactivation of Tris-inactivated chloroplasts

The light conditions required for reactivation of the oxygen-evolvingsystem in Tris-treated chloroplasts were studied by means ofrepetitive flashes. Inactive Tristreated chloroplasts were washedwith reduced 2,6-dichlorophenolindophenol, suspended in a reactivationmedium containing Mn²⁺⁺ and Ca²⁺⁺ ions, then illuminated withflashes. Flashes at dark intervals of 2 sec were most effectivefor reactivation, while those at shorter or longer intervalswere less effective. It was deduced that more than two sequentiallight reactions with dark reactions in between were involvedin the reactivation. (Received December 28, 1977; )     

The Photoreduction of Nicotinamide-Adenine Dinucleotide by Chromatophore Fractions from Rhodospirillum rubrum

The photoreduction of nicotinamide-adenine dinucleotide (NAD⁺), catalyzed by chromatophore fractions from young (1 day) and old (4-5 days) cultures of Rhodospirillum rubrum, was measured in the presence of either succinate or 2,6-dichlorophenol indophenol (DPIP) and an excess of ascorbate. The time-course of photoreduction in the succinate system suggested a “reversed electron flow” from the donor to NAD⁺ mediated by a high energy intermediate produced by a light-induced, cyclic electron transport in the chromatophore fractions. The effects of the uncoupler carbonyl cyanide [p-(trifluoromethoxy)phenyl]hydrazone (FCCP) and of the inhibitors antimycin A and 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO) were consistent with this interpretation. The time-course of NAD⁺ photoreduction in the presence of DPIP and ascorbate suggested a direct, light-induced electron transport from the donor to the acceptor. We cannot yet distinguish between a model in which the same reaction center is utilized in the photoreduction by both donor systems (the reaction center component P-870 may relate to two primary acceptors at different redox potential levels) and a model in which each photoreducing system is driven by its own reaction center component.     

Plastid Development in Primary Leaves of Phaseolus vulgaris: THE EFFECTS OF BRIEF FLASHES OF LIGHT ON DARK-GROWN PLANTS

Exposure of dark-grown beans to 1 ms flashes of light (2 ? 10¹⁴quanta/cm²/flash) at 15-min intervals induced growth of theprimary leaves as shown by increases in fresh weight, dry weight,and total protein. Effects of the flashes on plastid size andfine structure were not obvious until leaf growth was more thanhalf completed, when the prolamellar bodies became consumedand thylakoids were formed. Leaf samples taken after 638 and922 flashes contained some mesophyll cells with plastids ofabnormal appearance which had structures resembling stromacentrefibrils. Flashes of light increased both the chlorophyll content of theleaves and the activities seven enzymes of the photosyntheticcarbon cycle and of NAD-linked triosephosphate dehydrogenase(EC 1.2.1.12 [EC] ), these changes being correlated with leaf growthrather than the plastid changes detected by electron microscopy.There was only a small increase in the activity of phosphoribulokinase(EC 2.7.1.19 [EC] ) and no change in the activity of phosphopyruvatecarboxylase (EC 4.1.1.31 [EC] ).     

Die Dauer der Dunkeladaptation beim Fliegenauge nach Belichtung mit heterochromatischen Blitzen

Zusammenfassung Versuche mit heterochromatischen Lichtblitzen von 2 msec Dauer zeigen, daß die Dauer der Dunkeladaptation beim Calliphora-ERG von der Reihenfolge der Farbblitze abhängt. Versuche mit 2 diskreten Blitzabständen von 40 msec und 2 sec ergaben unterschiedliche Wirkungsspektren. Bei 40 msec Blitzabstand wirkt UV-Strahlung verlängernd auf die Adaptationszeit, bei 2 sec Abstand verkürzend. Die aufgenommenen Wirkungsspektren sprechen für das Auftreten eines kurzlebigen, bei 490 nm absorbierenden Intermediärfarbstoffes während der Potentialbildung und für das Auftreten eines stark im UV-Bereich absorbierenden Folgefarbstoffes sofort nach der Potentialbildung.

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Dark-adaptation after heterochromatic flash-illumination in the compound eye of the blow-fly

Summary The time course of dark adaptation (increase in amplitude of the electroretinogram) in the fly Calliphora was tested by light flashes (2 msec) of various wavelengths. The results showed that the time course depends on the wavelength of the test flashes, and on the interval between them. Using white test light the time for completion of dark adaptation increases with the interval between flashes, up to an interval of 20 sec. With longer intervals dark adaptation proceeds slightly faster. In contrast, using ultra-violet (UV) test light, a short (40 msec) interval increases the duration of the dark adaptation, and a longer (2 sec) interval reduces the duration. The results suggest that an intermediate photopigment is formed during the generation of the receptor potential. The pigment, which absorbs maximally at 490 nm, exists for a short period only. Immediately after the receptor potential is set up, an UV absorbing secondary pigment is formed.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft, SFB Bionach.     

Transformation of Protochlorophyllide, Formed from Exogenous δ-Aminolevulinic Acid, in Continuous Light and in Flashlight

δ-Aminolevulinic acid supplied to dark grown isolated leaves or wheat causes an accumulation of protochlorophyllide which is only partly transformed to chlorophyllide α in continuous light At the same time a considerable photodestruction of both pigments takes place. By a suitable combinations of short lights flashes and dark periods it is possible, however, to obtain at least double the amount of the protochlorophyllide transformed without photodestruction. The transformation isshown to be dependent on the dark interval between the light flashes. Possible connections with the formation of the protein part of the protochlorophyllide holochrome are discussed.     

Effects of manganese, calcium, dithiothreitol and bovine serum albumin on the light-reactivation of Tris-acetone-washed chloroplasts

Oxygen-evolution activity of spinach chloroplasts was investigatedby washing chloroplasts with 0.8M Tris buffer containing 20%acetone. This inactivitation was easily removed by two successivetreatments, dark- and light-reactivations. The first treatmentwas dark-reactivation step, rewashing inactivated chloroplastswith reduced DPIP (DPIP treatment). The second one was a light-reactivatedchloroplasts with incubating chloroplasts with Mn²⁺, Ca²⁺, dithiothreitoland bovine serum albumin under ilumination. Both light- and dark-reactivation treatments were required toregain oxygen-evolution activity of Tris-acetone-washed chloroplasts,which is characteristic of such chloroplasts. However, in Tris-washedchloroplasts considerable activity was recovered by dark-reactivationalone. Manganese and calcium contents of Tris-acetone-washed chloroplastswere compared with those of chloroplasts obtained by other preparations. Tris-acetone washing was presumed to inhibit the oxygen-evolutionsite of Photo-systetm II by affecting Mn, Ca and other substancesin chloroplasts. The inhibition site was estimated from a changein fluorescence yield of chlorophyll and the effect of artificialelectron donor specific for Photosystem II on NADP photoreductionactivity. (Received August 20, 1973; )     

Shifts to C675-670 and to C696-684 in etiolated leaves illuminated with series of brief flashes

Absorbance changes seen when etiolated primary bean leaves aregiven series of short, low-intensity flashes of light were recorded.Whatever its order in a series, each flash induced an initial,fast increase of the absorbance, producing an absorption bandbetween 660 and 700 nm with a peak at 680 nm. This occurredin less than 30 msec and was followed within seconds by eithera slow dark increase or a slow dark decrease of the leaf absorbance,depending on the order of the flash and the analytical wavelength.The dark absorbance changes were due to shifts of the initialabsorption band produced by the flash, either towards 685 or670 nm. The shift was always towards 670 nm after the firstflash. It appeared to move progressively towards 685 nm fromone flash to the next, this direction becoming predominant aftera certain number (n_inv) of flashes. The (n_inv) depended on thelight dose, but not on the flash frequency. Three proposalsare made to account for the results. (Received May 14, 1977; )     

Phytochrome-regulated growth of excised coleoptile tips of Triticum aestivum induced by blue, red and far-red light

The elongation growth of 5-mm tip sections of 3-day-old etiolatedwheat coleoptiles was promoted by light flashes of 436, 650and 750 nm in comparison with the dark control. As short as7/1,000 sec red light (R) of 14,420 W/m² led to saturation ofthe R-induced growth response. At quantum-identical far-redlight (FR) irradiation, a plateau developed at about 9/1,000sec which, however, was interrupted at longer irradiation periods.Varying dark periods between R or FR activation of growth anda second FR flash, a light-independent period with a half-lifeof 45 to 60 sec was found. Only after this light-independentperiod could R or FR activation of growth be reversed by FR.A second light-independent period was found by the followingirradiation schedule: 1 sec R/100 sec darknees(D)/l sec FR/varied D/l sec R. Our experimental conditions enabled us (a) to distinguish betweenactivation and inactivation by FR, (b) to clarify the reactionchain of different phytochrome forms and (c) to determine thehalf-life of light-independent phytochrome reactions.     

Activation of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase by Inorganic Phosphate under Nocturnal Conditions

In vivo activation states of ribulose 1,5-bisphosphate carboxylase/oxygenase(RuBisCO; EC 4.1.1.39 [EC] ) in the dark and light phases were measuredin intact leaves of Phaseolus and radish. The activation statewas high in the dark and comparable to the activation stateunder illumination at saturating light intensity. Then, we examined,using RuBisCO purified from spinach leaves, a mechanism forthe activation of RuBisCO in the dark when the stroma is neutralizedand lossess Mg²⁺ partly. Activation was not obserevd when theenzyme was incubated at air-level CO₂ and 10 mM Mg²⁺ at pH rangingfrom 6.2 to 7.5. However, the activation was highly promotedin this pH range when the activation mixture contained 10 mMinorganic phosphate. The activation state was 50 to 60% betweenpH 7.0 and 7.8 and maximum over pH 8.2 in the presence of 10mM inorganic phosphate. Studies of the initial rate of activationshow that the promotion of activation was through stabilizationof the active form of the enzyme by inorganic phosphate, notby altering the pKa of the activator -amino group of Lys-201.The physiological significance of the activation of RuBisCOby inorganic phosphate in the dark is discussed. ³ Present address: Department of Biochemistry, University ofNebraska, Lincoln, NE 68588-0664, U.S.A.     

Oxygen Evolution from Algae Illuminated by Short and Long Flashes of Light

The oxygen produced by illuminating Ankistrodesmus braunii withsingle light flashes has been determined using the Hersch galvanicoxygen cell. Measurements were made with the cells suspendedin alkaline solution equilibrated with nitrogen containing oxygenat a partial pressure of 10^–4 mm. Hg.

1. A single light flash, if very brief (less than 5 millisec.)results in no measurable oxygen production; a longer flash (35millisec.) gave a yield of approximately 1 mole O₂/800 moleschlorophyll.
2. A pair of flashes suitably spaced gave a greateryield thanthe sum of the yields when given individually, althoughonewas so brief that by itself it produced no measurable oxygen.The yield of a long flash preceded by a short flash was twiceas great as that of the long flash given alone; when the flashorder was reversed the combined yield was smaller but stillgreater than for the long flash alone.
3. The combined yieldof a pair of flashes varies with the intervalseparating theflashes, rapidly rising to a maximum and thendecaying moreslowly. With a long and short flash the optimalinterval was0.7 sec. but some enhancement of yield was observedwhen theflashes were separated by as long as 10 or 15 sec.
4. When theflashes were superimposed on background illuminationthe yieldswere increased and were measurable even for the shortflashes.Measured with background illumination the optimal yieldfora pair of short flashes was obtained with flashes separatedby about 0.05 sec.

     

COMPARATIVE STUDIES OF PHOTOCHEMICAL OXIDATION-REDUCTION REACTIONS IN LAMELLAR FRAGMENTS OF VARIOUS ALGAE AND SPINACH

The activity of various electron carriers, including DPIP, spinachplastocyanin, mammalian cytochrome c, and Anabaena cytochrome553, as donor in the reaction induced by the photochemical systemI was examined with lamellar fragments of various algae andspinach. Reduced DPIP was an effective electron donor irrespective ofthe organisms, when it was supplied at a high concentration(10^–3 M). Spinach plastocyanin was effective in the reactionswith the lamellae of green algae, Euglena, diatom Phaeodactyrumand red algae Porphyra yezoensis and Porphyra sp. Yamamoto II,whereas it was inactive in the lamellae of blue-green algae.Horse-heart cytochrome c and Anabaena cytochrome 553 were activein the reaction with the lamellae of bluegreen algae. The formercytochrome was also active in the reactions in Porphyridiumand Cyanidium. The cytochromes were less active in the reactionsin which spinach plastocyanin acted as effective electron donor. The data were interpreted as that the photochemical system Iin bluegreen algae differs from that of other photosyntheticorganisms with respect to the properties of the site of theelectron-input. ¹ Present address: Nomura Research Institute for Technologyand Economics, Kamakura, Kanagawa. ² Present address: Ocean Research Institute, University of Tokyo,Nakano, Tokyo.     

Afferent Stochastic Modulation of Crayfish Caudal Photoreceptor Units

When all roots to the sixth ganglion of the crayfish are cut, the caudal photoreceptor unit (PRU) fires at regular intervals. With an intact preparation, stimulation of caudal tactile hairs has predominantly inhibitory effects on the PRU: short bursts of afferent impulses, produced by momentary mechanical stimulation of tactile hairs, have (a) occasional immediate excitatory effect on the PRU, (b) prolonged inhibitory effect. The mean firing rate of the afferented and deafferented PRUs reacts similarly to a step increase in light, but the same unit fires faster after deafferentation. In the dark, deafferented units often fire paired or multiple pulses; the interval between pulses in a pair is similar to the short mode in afferented histograms. A fiber-optic probe of the caudal ganglion demonstrates the approximate location of the photosensitive element.     

Multiple-flash development of thermoluminescence bands in dark-grown spruce leaves

Spruce leaves greened in darkness were devoid of three of the five thermoluminescence bands found for mature leaves. These bands were developed rapidly by exposure of the dark-grown leaves to continuous light. The development of these bands was studied by illumination with repetitive flashes at varied intervals. Flashes at intervals of 1 s were the most effective in inducing these bands. Those at shorter or longer intervals were less effective. It was deduced from these data that the development of these bands is a multiquantum process which involves at least two photo-events with a dark reaction between them.     

Two modes of inhibition by 2-heptyl-4-hydroxyquinoline- N-oxide of electron transport associated with photosystem 2 in spinach chloroplasts

2-Heptyl-4-hydroxyquinoline-N-oxide (HOQNO) exerted two modesof inhibition on the Hill reaction in spinach chloroplasts.The first mode of inhibition by HOQNO, which developed rapidlyand reversibly on addition of the inhibitor to chloroplasts,was observed in the Hill reaction with various electron acceptorsand in photoreduction of methyl viologen with ascorbate as electrondonor, but was not observed in methyl viologen photoreductionsupported by reduced DPIP. Fifty percent inhibition of the Hillreaction was obtained with HOQNO at a low concentration of theorder of 10^–6 M. Besides the fast-developing inhibition, HOQNO caused a progressivedecrease in the Hill activity during the reaction when ferrricyanidewas used as electron acceptor. Similar time-dependent inactivationoccurred when chloroplasts were incubated with HOQNO and ferricyanidein the dark. Chloroplasts, thus inactivated, showed loweredactivity for ascorbate-supported methyl viologen photoreductionbut had unchanged activity for photoreduction with reduced DPIPas electron donor. Progress of the time-dependent inhibitionwas accelerated by increasing the concentration of HOQNO orferricyanide and was suppressed by addition of ferrocyanide.On changing the ratio of ferri- to ferrocyanide in the reactionmedium, fifty percent protection of the Hill activity was obtainedwith a ferri- and ferrocyanide mixture giving the redoxpotentialof 560 mv. A simple kinetic model for the second time-dependentinhibition by HOQNO is presented. ¹ Present address: Department of Botany, Faculty of Science,Toho University, Narashino 275, Chiba, Japan. (Received January 19, 1971; )     

Reactions of photosystems I and II in spinach chloroplast fragments treated with ethylene glycol

Photochemical reactions of chloroplast fragments isolated fromspinach leaves were measured in the presence of ethylene glycolor were measured after washing with an ethylene glycol-containingmedium. 2,6-Dichlorophenolindophenol (DPIP) photoreduction,oxygen evolution and oxygen uptake (a photosystem I reaction)were investigated in ethylene glycol-treated chloroplast fragments.By washing with ethylene glycol, oxygen evolution was stronglyinhibited, but oxygen uptake was not much affected by ethyleneglycol washing. Chloroplast fragments in 50% ethylene glycolmaintained a high rate of DPIP photoreduction (85% of the controlactivity in an ethylene glycol-less medium). In 67% ethyleneglycol, DPIP photoreduction mediated by photosystem II was eliminatedand only a small rapid reduction mediated by photosystem I wasobserved. Chloroplast fragments inhibited by ethylene glycolphotoreduced DPIP in the presence of p-aminophenol added asan artificial electron donor to photosystem II. The restoredactivity of DPIP photoreduction was inhibited by 3-(3',4'- dichlorophenyl)-1,1-dimethylurea. (Received September 8, 1970; )