Addition Of DCMU Research Articles

In situ autofluorescence detection of a fouling marine diatom on different surfaces

A new technique for quantifying fouling diatoms adhering to different surfaces was developed and tested. The method is based on recording the in vivo chlorophyll autofluorescence of diatom cells in situ. The enhanced signal obtained after addition of DCMU was used as a biomass estimate, and the enhancement itself as an indicator of the photosynthetic capacity. A fluorescence spectrometer equipped with a “well plate reader”; accessory was programmed to scan predetermined positions on the microscope slide based test surfaces. In standard tests, a matrix of 7 × 25 equidistant locations was applied to record the central 17 × 67 mm2 area of the test surface. Thus, both spatial distributions and the mean value of chlorophyll associated with the specified area could be obtained directly. Microscopical counting was performed for calibration on transparent glass surfaces as well as PVA‐SbQ based hydrogels. There was a good correlation between counting and fluorescence recordings, with a linear range up to 1100 cells mm−2. Due to the inherent inaccuracies of background estimates, the detection limit on glass and gel was approximately 200 cells mm−2. The method was also applied successfully to test non‐transparent surfaces. In addition to standard mass screening of different test surfaces, the method may be found useful in studies of algal physiology related to cell adhesion, photosynthesis, growth, detachment and spatial migration.

16 The role of amino acid residues in the S2 and S3 strands in the B β-sheet for the PAI-1 stability

In the glow curves of chloroplasts excited by a series of flashes at +1°C the intensity of the main thermoluminescence band appearing at +30°C (B band; B, secondary acceptor of Photosystem II) exhibits a period-4 oscillation with maxima on the 2nd and 6th flashes indicating the participation of the S3 state of the water-splitting system in the radiative charge recombination reaction. After long-term dark adaptation of chloroplasts (6 h), when the major part of the secondary acceptor pool (B pool) is oxidized, a period-2 contribution with maxima occurring at uneven flash numbers appears in the oscillation pattern. The B band can even be excited at −160°C as well as by a single flash in which case the water-splitting system undergoes only one transition (S1 → S2). The experimental observations and computer simulation of the oscillatory patterns suggest that the B band originates from charge recombination of the S2B− and S3B− redox states. The half-time of charge recombination responsible for the B band is 48 s. When a major part of the plastoquinone pool is reduced due to prolonged excitation of the chloroplasts by continuous light, a second band (Q band; Q, primary acceptor of Photosystem II) appears in the glow curve at +10°C which overlaps with the B band. In chloroplasts excited by flashes prior to DCMU addition only the Q band can be observed showing maxima in the oscillation pattern at flash numbers 2, 6 and 10. The Q band can also be induced by flashes after DCMU addition which allows only one transition of the water-splitting system (S1 → S2). In the presence of DCMU, electrons accumulate on the primary acceptor Q, thus the Q band can be ascribed to the charge recombination of either the S2Q− or S3Q− states depending on whether the water-splitting system is in the S2 or the S3 state. The half-time of the back reaction of Q− with the donor side of PS II (S2 or S3 states) is 3 s. It was also observed that in a sequence of flashes the peak positions of the Q and B bands do not depend on the advancement of the water-splitting system from the S2 state to the S3 state. This result implies that the midpoint potential of the water-splitting system remains unmodified during the S2 → S3 transition.

Heterogeneity and photoinhibition of photosystem II studied with thermoluminescence

Thermoluminescence (TL) signals were recorded from grana stacks, margins, and stroma lamellae from fractionated, dark-adapted thylakoid membranes of spinach (Spinacia oleracea L.) in the absence and in the presence of 2,6-dichlorphenylindophenol (DCMU). In the absence of DCMU, the TL signal from grana fractions consisted of a homogenous B-band, which originates from recombination of the semi-quinone QB- with the S2 state of the water-splitting complex and reflects active photosystem II (PSII). In the presence of DCMU, the B-band was replaced by the Q-band, which originates from an S2QA- recombination. Margin fractions mainly showed two TL-bands, the B- and C-bands, at approximately 50 degreesC in the absence of DCMU, and Q- and C-bands in the presence of DCMU. The C-band is ascribed to a TyrD+-QA- recombination. In the absence of DCMU, the fractions of stromal lamellae mainly gave rise to a TL emission at 42 degreesC. The intensity of this band was independent of the number of excitation flashes and was shifted to higher temperatures (52 degreesC) after the addition of DCMU. Based on these observations, this band was considered to be a C-band. After photoinhibitory light treatment of uncoupled thylakoid membranes, the TL intensities of the B- and Q-bands decreased, whereas the intensity at 45 degreesC (C-band) slightly increased. It is proposed that the 42 to 52 degreesC band that was observed in marginal and stromal lamellae and in photoinhibited thylakoid membranes reflects inactive PSII centers that are assumed to be equivalent to inactive PSII QB-nonreducing centers.

Effect of carbon and nitrogen availability on intracellular amino acids and ammonium pools in the green alga Monoraphidium braunii

In this paper, we present results of our studies on nitrogen metabolism in the green alga Monoraphidium braunii , strain 202–7 d, concerning changes in intracellular ammonium, glutamate and glutamine pools (as determined by reversed phase chromatography). We found that blocking of nitrogen assimilation at the GS or GOGAT steps quickly induced changes in intracellular amino acid levels, which were opposite in the case of Gln. Monoraphidium cultures can recover from the effect of such inhibitors, probably by its metabolization. Darkness or addition of DCMU affected GS regulation in the same way in Monoraphidium , but they provoked distinct effects on intracellular pools of amino acids. The inhibition of GS activity had a similar effect on the levels of primary products of ammonium assimilation (Glu and Gln) regardless of the energetic state of cells.

Chlorophyll organization in dark‐grown and light‐grown pine (Pinus brutia) and barley (Hordeum vulgare)

A study was conducted comparing the organization of chlorophyll during development of the photosynthetic apparatus in dark‐grown and light‐grown pine and barley. The rationale was that gymnosperms, but not angiosperms, have a capacity to synthesize chlorophyll in darkness. Seedlings of Pinus brutia were germinated and grown in darkness or under photoperiodic (day/night) conditions. The low‐temperature (77 K) fluorescence spectra of newly‐emerging dark‐grown seedlings exhibited a single fluorescence band peaking at 678–679 nm, which decayed primarily with a ∼5.5 ns lifetime. Over the first few days of growth, the emission shifted to longer wavelengths and a subnanosecond lifetime component became prevalent. After several days of dark growth the emission spectrum and lifetime profile of the low temperature fluorescence came to resemble those of light‐grown pine and barley. At room temperature, dark‐grown pine showed little variable fluorescence, though addition of DCMU caused a substantial fluorescence rise. Illumination with moderate light for a few hours was sufficient to ‘photoinduce’ the appearance of normal variable fluorescence. At 77 K, DCMU‐treated samples clearly showed a very long‐lived (∼40 ns) fluorescence lifetime component in light‐grown pine and barley. This component was undetectable in dark‐grown pine. If, however, dark‐grown samples were illuminated either before or after DCMU addition and then frozen to 77 K, the ∼40 ns lifetime component appeared at a fluorescence intensity similar to that in light‐grown samples. These results are explained primarily in terms of photoactivation of the photosystem II (PSII) donor side. The temporary maintenance of PSII in an inactive, highly‐quenched state is suggested to provide an available, yet protected precursor for active PSII.

A time-resolved FTIR difference study of the plastoquinone QA and redox-active tyrosine YZ interactions in photosystem II.

In this paper, we present the first time-dependent measurements of flash-induced infrared difference spectra of photosystem II (PSII) using Fourier transform infrared (FTIR) spectroscopy. With this experimental approach, we were able to obtain the YZoxQA-/YZQA vibrational difference spectrum of Tris-washed, PSII-enriched samples in the absence of hydroxylamine at room temperature (16 +/- 2 degrees C), with a spectral resolution of 4 cm-1 and a temporal resolution of 50 ms. In order to determine the dominant species in the FTIR spectrum at a particular point in time after an excitation flash, the decay kinetics of YZox and QA- were independently monitored by EPR and chlorophyll a fluorescence, respectively, under the same experimental conditions. These measurements confirmed that the addition of DCMU to Tris-washed PSII samples does not significantly affect the YZox decay, but does substantially slow down the QA- decay. By making use of the difference in the decay kinetics using DCMU, the QA-/QA signals could be separated from the YZox/YZ signals and a pure QA-/QA difference spectrum obtained. By comparison of the YZoxQA-/YZQA difference spectrum with the pure QA-/QA difference spectrum, a large differential band at 1706/1699 cm-1 could be identified and associated with YZ oxidation. In contrast, an intense band at 1478 cm-1, whose DCMU-sensitive decay follows the QA- decay based on the chlorophyll a fluorescence measurements, was present in all of the time-resolved spectra. Since no significant reversible Chl+ radicals could be detected by the EPR measurements under our experimental conditions, we confirm that this band most likely arises only from the semiquinone anion QA- [Berthomieu, C., Nabedryk, E., Mäntele, W., & Breton, J. (1990) FEBS Lett. 269, 363-367].

Characterization of the non-photochemical quenching of chlorophyll fluorescence that occurs during the active accumulation of inorganic carbon in the cyanobacterium Synechococcus PCC 7942

Previous work has shown that the maximum fluorescence yield from PS 2 of Synechococcus PCC 7942 occurs when the cells are at the CO2 compensation point. The addition of inorganic carbon (Ci), as CO2 or HCO3 (-), causes a lowering of the fluorescence yield due to both photochemical (qp) and non-photochemical (qN) quenching. In this paper, we characterize the qN that is induced by Ci addition to cells grown at high light intensities (500 μmol photons m(-2) s(-1)). The Ci-induced qN was considerably greater in these cells than in cells grown at low light intensities (50 μmol photons m(-2) s(-1)), when assayed at a white light (WL) intensity of 250 μmol photons m(-2) s(-1). In high-light grown cells we measured qN values as high as 70%, while in low-light grown cells the qN was about 16%. The qN was relieved when cells regained the CO2 compensation point, when cells were illuminated by supplemental far-red light (FRL) absorbed mainly by PS 1, or when cells were illuminated with increased WL intensities. These characteristics indicate that the qN was not a form of energy quenching (qE). Supplemental FRL illumination caused significant enhancement of photosynthetic O2 evolution that could be correlated with the changes in qp and qN. The increases in qp induced by Ci addition represent increases in the effective quantum yield of PS 2 due to increased levels of oxidized QA. The increase in qN induced by Ci represents a decrease in PS 2 activity related to decreases in the potential quantum yield. The lack of diagnostic changes in the 77 K fluorescence emission spectrum argue against qN being related to classical state transitions, in which the decrease in potential quantum yield of PS 2 is due either to a decrease in absorption cross-section or by increased 'spill-over' of excitation energy to PS 1. Both the Ci-induced qp (t 0.5<0.5 s) and qN (t 0.5≃1.6 s) were rapidly relieved by the addition of DCMU. The two time constants give further support for two separate quenching mechanisms. We have thus characterized a novel form of qN in cyanobacteria, not related to state transitions or energy quenching, which is induced by the addition of Ci to cells at the CO2-compensation point.

Alteration in the acceptor side of photosystem II of chloroplast by high light

The effect of high light on the acceptor side of photosystem II of chloroplasts and core particles of spinach was studied. BothVmax and apparentKm for DCIP were altered in photoinhibited photosystem II core particles. The double reciprocal plot analysis as a function of actinic light showed increased slope in chloroplasts photoinhibited in the presence of DCMU. Exposure of chloroplasts to high light in the presence of DCMU did not protect the chloroplast against high light induced decrease in Fm, level. Further the high light stress induced decrease inFm level was not restored by the addition of DCMU. These results suggest that the high light stress induced damage to chloroplast involves alteration in the binding site forQB on the DI protein on the acceptor side of photosystem II

Interaction of the photosynthetic and respiratory electron transport chains producing slow O2 signals under flashing light in Synechocystis sp. PCC 6803

We investigated the slow signal of apparent O2 release under brief light flashes by using mutants of Synechocystis sp. PCC 6803 which lacked CP43 and D1. The slow signal was present at higher amplitudes in the mutants. It was inhibited by starving the mutants of glucose (>90%), by 10 mM NaN3 (85%) and by boiling samples for 2 min (100%). In the mutants and in the wild-type, the slow signal was 95% inhibited by the combination of DBMIB (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone) and HQNO (2-n-heptyl-4-hydroxyquinoline-N-oxide). In the wild type, the addition of DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) or CCCP (carbonylcyanide m-chlorophenylhydrazone) completely inhibited photosynthetic O2 evolution, yet failed to inhibit the slow signal. We explain the kinetics of the wild-type signal as a positive deflection due to the inhibition of respiration by PS I activity, and a negative deflection due to the stimulation of respiration by electrons originating from PS II. We found no evidence of a 'meta-stable S3' in Synechocystis sp. PCC 6803 that could contribute to the slow signal of apparent O2 release. We present a calculation which involves only averaging, division and subtraction, that can remove the contribution of the slow signal from the true photosynthetic O2 signal and provide up to a 10-fold improved accuracy of the S-state models.

Assessing modulation of stromal and thylakoid light-harvesting complex-II phosphatase activities with phosphopeptide substrates

The study of the light-harvesting complex II (LHC-II) phosphatase activity has been difficult due to the membrane association of its substrate. Thylakoid membranes labeled with [γ-(32)P]ATP were incubated with chymotrypsin, releasing phosphopeptides which served as labeled substrates for LHC-II phosphatase. Utilizing these phosphopeptides as substrates, protein phosphatase activities have been identified in both the thylakoid membrane and the stromal fraction. The thylakoid-bound phosphatase was liberated from the membrane with a sub-solubilizing concentration of Brij 35. The membrane and the stromal protein phosphatases were inhibited by NaF and EDTA, but not inhibited by microcystin-LR. The stromal phosphatase differed from the membrane phosphatase in pH optimum, in its lack of inhibition by molybdate ions, and by its response to magnesium and manganese ions. Using the soluble chymotryptic peptide substrate, the effect of light on pea thylakoid-bound LHC-II phosphatase activity was also assessed. Incubation of the thylakoid membranes in the light caused a 35% inhibition of LHC-II phosphatase activity. The inhibition was diminished by the addition of DCMU. Addition of 10 mM dithiothreitol stimulated the activity in darkness and obviated the inhibition when exposed to light. These studies suggest that positive or negative regulation of the LHC-II phosphatase activity is possible in vivo.

Nitrogen fixation and hydrogen uptake in four cyanobacteria

Anabaena variabilis, Nostoc spongiaeforme, Westiellopsis prolifica, and Nostoc sp. isolated from diverse local habitats fixed nitrogen under aerobic conditions, but the nitrogenase activity was greater in anaerobic conditions. Nitrogenase activity was highest in fluorescent light in the case of A. variabilis, W. prolifica and Nostoc sp., but that of N. spongiaeforme was higher in incandescent light. N. spongiaeforme showed high nitrogenase activity in blue light also, whereas the other three species had stronger activity in red light than in blue light. A concentration of H 2 up to 20% enhanced the nitrogenase activity, but 25% was inhibitory in all of the four organisms. All the four strains were found capable of utilizing exogenous hydrogen. N. spongiaeforme showed higher H 2 uptake activity both under aerobic and anaerobic conditions and its capacity for chromatic adaptation may partly account for such behaviour. Addition of DCMU inhibited hydrogen uptake in A. variabilis, W. prolifica and Nostoc sp. but not in N. spongiaeforme.

Ecophysiology of stromatolitic microbial mats, Stocking Island, exuma cays, Bahamas

Intertidal stromatolites, covered by cyanobacterial mats, were recently discovered at Stocking Island, Exuma Cays, Bahamas. Ecophysiological responses (CO2 fixation, N2 fixation, and photoacclimation) of these cyanobacterial mats to experimental manipulations were examined to identify potential environmental variables controlling community structure and function. The mats exhibit horizontal zonation that shifts from soft to crusty to hard in a seaward direction. Cluster analysis of chemotaxonomic photopigments (chlorophylls and carotenoids) revealed that visually distinct mat types are composed of distinct phototrophic assemblages. Under reduced irradiance, diatoms within the mats photoacclimated by increasing accessory photopigments (diadinoxanthin, fucoxanthin, and chlorophyll c 1 c 2) and cyanobacteria reduced the photoprotective carotenoid echinenone. In a 4-day nutrient addition bioassay experiment, nitrate, phosphate, dissolved organic carbon, and trace metal enrichments did not enhance CO2 fixation, but phosphate enrichments tripled N2 fixation rates. The addition of DCMU increased N2 fixation rates relative to nonamended light and dark rates, indicating light (photosystem I) enhanced nitrogenase activity. Soft mats appear to represent the early stages of colonization and stabilization of mat communities. Active growth following stabilization results in the formation of partially-lithified crusty mats, which eventually become highly-lithified and form hard mats. Collectively, our results suggest that Stocking Island stromatolitic mats have low growth rates and consequently exhibit slow responses to increased nutrient availability and changes in ambient irradiance. In general, intertidal stromatolitic mats at Stocking Island appear to exhibit low rates of CO2 and N2 fixation relative to nonlithifying temperate cyanobacteral mats. Although production is low, respiration is likewise low, leading to the suggestion that high production to respiration ratios (P:R) may be necessary for lithification of intertidal stromatolitic mats.

BIO‐OPTICAL CHARACTERISTICS AND PHOTOADAPTIVE RESPONSES IN THE TOXIC AND BLOOM‐FORMING DINOFLAGELLATES GYRODINIUM AUREOLUM, GYMNODINIUM GALATHEANUM, AND TWO STRAINS OF PROROCENTRUM MINIMUM1

ABSTRACTPhotoadaptive responses in the toxic and bloom‐forming dinoflagellates Gyrodinium aureolum Hulbert, Gymnodinium galatheanum Braarud, and two strains of Prorocentrum minimum (Pavillard)Schiller were evaluated with respect to pigment composition, light‐harvesting characteristics, carbon and nitrogen contents, and growth rates in shade‐ and light‐adapted cells. The two former species were grown at scalar irradiances of 30 and 170 μmol · m −2 at a 12‐h daylength at 20° C. The two strains of P. minimum were grown at 35 and 500 μmol. m−2· s−1 at a 2‐h daylength at 20° C. For the first time, chlorophyll (chl) c3, characteristic of several bloom‐forming prymnesiophytes, was detected in G. aureolum and G. galatheanum. Photoadaptional status affected the pigment composition strongly, and the interpretation of the variation depended on whether the pigment composition was normalized per cell, carbon, or chl a. Species‐specific and photoadaptional differences in chl a‐specific absorption (°ac, 400–700 nm) and chl a‐normalized fluorescence excitation spectra of photosystem II fluorescence with or without addition of DCMU (°F and °FDCMU 400–700 nm) were evident. Gyrodinium aureolum and G. galatheanum exhibited in vivo spectral characteristics similar to chl c3‐containing prymnesiophytes in accordance with their similar pigmentation. Prorocentrum minimum had in vivo absorption and fluorescence characteristics typical for peridinin‐containing dinoflagellates. Species‐specific differences in in vivo absorption were also observed as a function of package effect vs. growth irradiance. This effect could be explained by differences in intracellular pigment content, cell size/shape, and chloroplast morphology/numbers. Light‐ and shade‐adapted cells of P. minimum contained 43 and 17% of photoprotective carotenoids (diadino + diatoxanthin) relative to chl a, respectively. The photoprotective function of these carotenoids was clearly observed as a reduction in °F and °F DCMU at 400–540 nm compared to °ac in light‐adapted cells of P. minimum. Spectrally weighted light absorption (normalized to chl a and carbon, 400–700 nm) varied with species and growth conditions. The use of quantum‐corrected and normalized fluorescence excitation spectra with or without DCMU‐treated cells to estimate photosynthetically usable light is discussed. The usefulness of in vitro absorption and fluorescence excitation spectra for estimation of the degradation status of chl a and the ratio of chl a to total pigments is also discussed.

Low-pH-induced Ca 2+ ion release in the water-splitting system is accompanied by a shift in the midpoint redox potential of the primary quinone acceptor Q A

pH-dependent regulation of Photosystem (PS) II (observed as ‘high-energy quenching’) has been characterized by chlorophyll fluorescence and thermoluminescence measurements in PS II particles, thylakoid membranes, alga cells and leaf tissue. Steady state redox titration of fluorescence yield performed at pH 6.5 revealed that the midpoint redox potential of the primary quinone acceptor, Q A, is shifted towards positive direction from E m = −80 mV to E m = +40 mV (the absolute values for E m were varying by about 40 mV between different preparations) after incubation of PS II particles at pH 4.2 for 15 min in the presence of the Ca 2+ chelator, PP i. The original midpoint potential was restored after the addition of 300 μM CaCl 2. Low-pH treatment (pH 4.6) of PS II particles also resulted in a decrease of the Q band of thermoluminescence (appearing between 10–14°C after DCMU addition) with a concomitant appearance or intensification of a high temperature band between 42–50°C (C band). In accordance with the results of the redox titration of fluorescence yield the C band is attributed to a low-pH-induced high potential form of Q A. The interconversion of Q band into the C band was more pronounced in the presence of the Ca 2+ chelator, EGTA. Addition of CaCl 2 to the low-pH-treated particles diminished the C band and restored the Q band. Light-induced acidification of the thylakoid lumen (ΔpH formation under illumination conditions of ‘high-energy quenching’) was also accompanied by a transformation of the Q band to the C band in isolated thylakoids, in the green alga, Chlorella vulgaris and in pea leaves. The phenomenon was completely reversed by abolishing the pH gradient with 10 mM NH 4Cl. Addition of the Ca 2+-channel inhibitor verapamil to the thylakoid suspension before the formation of a ΔpH suppressed the transformation of Q band into the C band. In contrast, when a ΔpH was first established and then verapamil was added, the ΔpH-induced change in the glow curve was irreversible and conversion of C band back to the Q band was prevented. It is suggested that the appearance of the C band is associated with Ca 2+-dependent reversible inactivation of the water-splitting system and with a shift in the redox potential of Q A. We propose that pH-dependent Ca 2+-release is a physiological process which controls the electron transport of PS II in vivo.

Diurnal nitrogen fixation in tropical marine cyanobacteria: a comparison between adjacent communities of non-heterocystousLyngbyasp. and heterocystousCalothrixsp.

Nitrogenase activity (ARA) in adjacent marine tropical communities of a non-heterocystous Lyngbya sp. and a heterocystous Calothrix sp. was investigated in the light and the dark. For both cyanobacteria, in situ ARA increased throughout the day except for afternoon reductions during cloud cover. ARA rates varied on different days but Calothrix sp. was always more active during daylight hours than Lyngbya sp. Both cyanobacteria showed only very low rates of ARA when incubated in the dark in situ during normal daylight hours. Nocturnal ARA, at 82% the daylight rate in Lyngbya sp., and 18% the daylight rate in Calothrix sp., was three times higher in Lyngbya sp. than in Calothrix sp. In both, nocturnal ARA rose from a low rate after dusk to a peak around midnight and declined towards dawn. In each organism ARA in the light was higher than in the dark regardless of the time of day. The addition of DCMU, an inhibitor of photosystem II, generally inhibited nitrogenase activity in the heterocystous Calothrix sp....

Investigation of the origin of the "S3" EPR signal from the oxygen-evolving complex of photosystem 2: the role of tyrosine Z.

The origin of the "S3" EPR signal from calcium-depleted photosystem 2 samples has been investigated. This signal is observed after freezing samples under illumination and has been assigned to an interaction between the manganese cluster and an oxidized histidine radical [Boussac et al. (1990) Nature 347; 303-306]. In calcium-depleted samples prepared by three different methods, we observed the trapping of the tyrosine radical YZ+ under conditions which also formed the "S3" signal. An "S3"-type signal and YZ+ were also formed in PS2 samples treated with the water analogue ammonia. Following illumination at 277 K, the "S3" and YZ+ signals decayed at the same rate at 273 K in the dark. Both the YZ+ and "S3" signals decayed on storage at 77 K and could be subsequently regenerated by illumination at 8-77 K. No evidence to support histidine oxidation was found. The effects of DCMU, chelators, and alkaline pH on the dark-stable multiline S2 and the "S3" signals from calcium-depleted samples were determined. Both signals required the presence of EGTA or citrate for maximum yield. The addition of DCMU caused a reduction in the yield of "S3" generated by freezing under illumination. Incubation at pH 7.5 resulted in the loss of both signals. We propose that a variety of treatments which affect calcium and chloride binding cause a stabilization of the S2 state and slow the reduction of YZ+. This allows the trapping of YZ+, the interaction with the manganese cluster (probably in the S2 state) resulting in the "S3" signal. The data allow the position of the manganese cluster to be estimated as within 10 A of tyrosine Z (D1-161).

Two Distinct Modes of Action of Light on the in vitro Development of Root Explants of Cichorium intybus L.

Summary Modalities of action of light on the in vitro development of endive ( Cichorium intybus L.) root tissues were examined by comparing the effects of two photoperiodic conditions (i.e. SD of 9 h light, 5.6 Wm -2 , and LD of 16 h light with equal wattage). The influence of LD and SD was first studied on the structural events that take place from the beginning target cells until the appearance of regenerated buds. In both LD and SD conditions, the typical elements of a cellular dedifferentiation can be detected but LD supply resulted in the formation of more buds that also emerged more rapidly than those in SD. The increase of light intensity to 10Wm -2 or the exogenous supply of sucrose (2–5%) in SD conditions enhanced the amount of the newly formed buds in comparison with controls, whereas the addition of DCMU to the culture medium in LD partially inhibited bud regeneration, 5.10 -6 M DCMU being the most effective concentration. After day 10 of the culture, the mitotic activity was given major attention to characterize the functioning of the newly formed buds. LD induced a progressively prefloral stage, the culmination of which was generally achieved on day 16. In SD conditions, some (20%) intermediate meristems could be observed with some delay (on day 24), but flowering was never completed, even after transfer to LD. Increasing the quantity of light in SD to an equivalent LD level resulted in a significant rise in the mitotic activity in the peripheral zone only; consequently, the zonation was retained. It is concluded that light acts first as an energizing source to control the bud's «setting-up» from the start to day 10 of the culture; secondly, light intervenes as an informative source to determine the differentiation, vegetative or reproductive, of the newly formed buds.

Secondary electron transfer reactions of the isolated Photosystem II reaction centre after reconstitution with plastoquinone-9 and diacylglycerolipids

A Photosystem II (PS II) reaction centre preparation isolated with Triton X-100 and stabilised by transfer to dodecylmaltoside has been used in a reconstitution procedure with diacyl lipids and the naturally occurring PS II quinone, plastoquinone-9 (PQ9). As shown previously, this isolated protein complex consists of the D1, D2, b -559 α and β polypeptides, plus the psb I gene product. Before reconstitution there was only about 0.004 mol PQ9 and 0.2 mol diacyl lipid per mole chlorophyll a associated with the isolated complex. After reconstitution and precipitation by centrifugation the complex was found to have an associated lipid matrix of 1.3 mol PQ9 and 3.7 mol diacyl lipid for each mole of chlorophyll a . After reconstitution, the presence of a functional quinone in the PS II reaction centre was indicated by a strong thermoluminescence signal with a peak emission at −60°C. This was recognised as the previously characterised Z v signal on the basis of the relationship between emission and excitation temperatures and a complete quenching by ethanol. This signal was essentially unaltered by addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and was the same after reconstitution with phosphatidylcholine or a thylakoid lipid extract. Calculation of the activation free energy for the charge separation reaction which gave rise to this signal suggested that this was 0.48 ± 0.01 eV and was the same as for a smaller Z v signal detected in the isolated complex before reconstitution with PQ9. When duroquinone was used this value was not significantly different, although a much smaller signal was observed. With decylplastoquinone the thermoluminescence peak was as large as with PQ9 but the difference in shape indicated that the activation free energy was significantly higher at 0.55 ± 0.02 eV. Photosynthetic electron transfer in the reconstituted complex was assayed by reduction of 2,6-dichlorophenolindophenol (DCPIP) with diphenylcarbazide (DPC) as an artificial electron donor. A low residual rate was strongly enhanced by reconstitution with PQ9. This activity was greater when reconstitution was with a thylakoid lipid extract than with phosphatidylcholine and was inhibited by addition of DCMU ( I 50 = 0.2 mM).

Determination of nitrate reductase activity in Ulva rigida C. Agardh by the in situ method

Assay conditions for determining in situ nitrate reductase (NR) activity in Ulva rigida C. Agardh were studied. The final composition of the incubation medium was: 0.1 M phosphate buffer, 0.5 mM EDTA, 0.1% 1-propanol, 30 mM KNO 3 and 10 μM glucose. The optimal pH was 8, which is greater than that usually found in higher plants (7.5) and close to the pH of sea water. Samples were incubated in the dark at 30 °C. A short incubation time (30 min) was more satisfactory than longer ones for determination of the initial rate, and to prevent any limitation of the NO 3 − reduction rate for the assay conditions themselves. Surfactants such as Tergitol NP-10 and Triton X-100 were less effective than 0.1% 1-propanol. Incubation in anaerobic conditions is a critical point in the in situ NR assay in Ulva rigida. The two methods used; vacuum infiltration for 10 min, and N 2-flushing 2 min before and 2 min after introducing the sample of tissue, were almost identically effective. The addition of NADH and glucose as external sources of reducing power was studied and the problems associated with NADH interference in NO 2 − determination are discussed. The addition of DCMU did not overcome the need for darkness as it does in higher plants, even when the photosynthetic O 2-production was completely abolished at the same DCMU concentration.

Non-photochemical quenching of chlorophyll fluorescence in the green alga Dunaliella

The relaxation of the non-photochemical quenching of chlorophyll fluorescence has been investigated in cells of the green alga Dunaliella following illumination. The relaxation after the addition of DCMU or darkening was strongly biphasic. The uncoupler NH4Cl induced rapid relaxation of both phases, which were therefore both energy-dependent quenching, qE. The proportion of the slow phase of qE increased at increasing light intensity. In the presence of the inhibitors rotenone and antimycin the slow phase of qE was stabilised for in excess of 15 min. NaN3 inhibited the relaxation of almost all the qE. The implications of these results are discussed in terms of the interpretation of the non-photochemical quenching of chlorophyll fluorescence in vivo and the mechanism of qE.