Changes In Fluorescence Yield Research Articles

Changes of Chlorophyll(ide) Fluorescence Yield Induced by a Short Light Pulse as a Probe to Monitor the Early Steps of Etioplast Phototransformation in Dark-Grown Leaves

Changes of Chlorophyll(ide) Fluorescence Yield Induced by a Short Light Pulse as a Probe to Monitor the Early Steps of Etioplast Phototransformation in Dark-Grown Leaves

Changes of Chlorophyll(ide) Fluorescence Yield Induced by a Short Light Pulse as a Probe to Monitor the Early Steps of Etioplast Phototransformation in Dark-Grown Leaves

The fluorescence yield of chlorophyll(ide) (Chl[ide]) excited by weak modulated light was recorded at room temperature during a 2 h period after a short actinic light pulse that transformed all photoactive protochlorophyllide in dark-grown barley leaves. A typical pattern of fluorescence yield variations was found whatever the age of the leaf but with age-dependent changes in rates. Its successive phases were related to the Chl(ide) spectral shifts observed in low-temperature emission spectra. The fluorescence yield started at a high level and strongly declined during the formation of Chlide695 from Chlide668 within a few seconds. It increased to a transient maximum during the Shibata shift (15–25 min) that resulted in Chl(ide)682. A final, slow decrease to a steady state occurred during the final red shift to Chl685. Pretreatments with δ-aminolevulinic acid, chloramphenicol or 1, 10-phenanthroline resulted in correlated modifications of Chl(ide) fluorescence yield transients and shifts of the low-temperature Chl(ide) emission band. The complex response of the final decrease phase of the fluorescence yield to these compounds suggests that it results both from the assembly of photosynthetic Chl proteins and from the reorganization of the etioplast membrane system. From these results it is concluded that continuous recordings of Chl(ide) fluorescence yield after a short light pulse represent a useful tool to monitor the kinetics of pigment–protein organization and primary thylakoid assembly triggered by Pchlide photoreduction.

Bicarbonate protects the donor side of photosystem II against photoinhibition and thermoinactivation

The rate of photoinhibition of photosystem II (PSII) activities (photoinduced change of chlorophyll fluorescence yield, ΔF, and photoreduction of 2,6-dichlorophenol-indophenol) in O 2-evolving pea subchloroplast membrane fragments in medium depleted of CO 2 was considerably decreased upon addition of 5 mM NaHCO 3 before the light treatment. A similar effect was observed when 100 μM MnCl 2 was added before the photoinhibition. In PSII membrane fragments depleted of Mn, the pre-illumination led to irreversible loss of the capability of PSII to be reactivated by Mn 2+, and the rate of the photoinhibition was decreased by a factor of 2 or 5 if the pre-illumination was done in the presence of 0.2 μM MnCl 2 (≈4 Mn per PSII reaction center) added alone or in combination with 5 mM NaHCO 3, respectively. A similar protective effect of bicarbonate was also revealed in the dark, during thermoinactivation of O 2-evolving PSII at 40°C: the rate of thermoinactivation of ΔF was decreased by a factor of 3 if 5 mM NaHCO 3 was added to the medium. The results are consistent with the idea that bicarbonate is an essential component of the Mn-containing water-oxidizing complex of PSII, which decreases its susceptibility to photoinhibition and thermoinactivation.

Oxygen dependence of photosynthetic electron transport in a bacteriochlorophyll-containing rhizobium

Bacteriochlorophyll-containing rhizobia, which form nitrogen-fixing nodules on the stems and roots of the legume Aeschynomene, grow photosynthetically only in the presence of oxygen or auxiliary electron acceptors. We show that, in whole cells of the Rhizobium strain BTAi 1, a single-turnover excitation flash photooxidized c-type cytochrome under aerobic but not anaerobic conditions. Light-induced fluorescence yield changes show that under anaerobic conditions, the primary acceptor quinone, Q A, is predominantly in the reduced state and so unable to accept electrons. Thus, as is the case for the aerobic photosynthetic bacterium Roseobacter denitrificans, over-reduction of Q A likely prohibits photosynthesis under anaerobic conditions.

On the Requirement of Bound Bicarbonate for Photosystem II Activity

Abstract In photosystem II of plants and cyanobacteria, but not in reaction centers of anoxygenic photosynthetic bacteria, formate is known to inhibit electron flow which is reversed fully upon bicarbonate addition. At issue has been an old controversy whether this effect is on the acceptor or the donor side of photosystem II (PS II). We present here data on chloroplast thylakoids for donor side effects, that is accompanied by acceptor side effects, from measurements on chlorophyll a fluorescence yield changes after light flashes 1-6. Further, sensitive differential infrared gas analyser measurements show that bicarbonate is indeed bound in both maize and pea thylakoid suspensions depleted of CO2 without any inhibitor; here, high rates of electron flow are associated with the presence of a maximum of 0.8 to 1.25 (corrected for residual activity) CO2 per photosystem II reaction center. It is suggested that bicarbonate bound to the acceptor side is required for photosystem II activity , both on the acceptor and the donor sides in the same experiment and in the same sample.

Electron transfer in reaction centers of Rhodobacter sphaeroides and Rhodobacter capsulatus monitored by fluorescence of the bacteriochlorophyll dimer

Spectral and kinetic characteristics of fluorescence from isolated reaction centers of photosynthetic purple bacteria Rhodobacter sphaeroides and Rhodobacter capsulatus were measured at room temperature under rectangular shape of excitation at 810 nm. The kinetics of fluorescence at 915 nm reflected redox changes due to light and dark reactions in the donor and acceptor quinone complex of the reaction center as identified by absorption changes at 865 nm (bacteriochlorophyll dimer) and 450 nm (quinones) measured simultaneously with the fluorescence. Based on redox titration and gradual bleaching of the dimer, the yield of fluorescence from reaction centers could be separated into a time-dependent (originating from the dimer) and a constant part (coming from contaminating pigment (detached bacteriochlorin)). The origin was also confirmed by the corresponding excitation spectra of the 915 nm fluorescence. The ratio of yields of constant fluorescence over variable fluorescence was much smaller in Rhodobacter sphaeroides (0.15±0.1) than in Rhodobacter capsulatus (1.2±0.3). It was shown that the changes in fluorescence yield reflected the disappearance of the dimer and the quenching by the oxidized primary quinone. The redox changes of the secondary quinone did not have any influence on the yield but excess quinone in the solution quenched the (constant part of) fluorescence. The relative yields of fluorescence in different redox states of the reaction center were tabulated. The fluorescence of the dimer can be used as an effective tool in studies of redox reactions in reaction centers, an alternative to the measurements of absorption kinetics.

Characteristic changes of function and structure of Photosystem II during strong-light photoinhibition under aerobic conditions

The effects of strong-light photoinhibition under aerobic condition on the function and structure of Photosystem (PS) II were studied by measuring flash-induced changes of chlorophyll fluorescence yield, thermoluminescence glow curves, Fourier transform infrared (FTIR) spectra and the amounts of the PS II proteins, including the D1 and D2 proteins, in PS II membranes. Aerobic photoinhibition resulted in decrease in light-induced FTIR difference spectra of PS II. The intensity of the positive band at 1479 cm −1 due to a semiquinone anion in the Q A pocket decreased with the same kinetics as the inhibition of O 2-evolving activity. In an early stage of photoinhibition, the very fast decaying component of flash-induced fluorescence relaxation due to rapid electron transfer from Q A − to Q B was eliminated, and the intensity of thermoluminescence band due to S 2Q B − charge recombination decreased concomitant with appearance of another band due to S 2Q A − charge recombination. These results suggested that the impairment of Q B function preceded the loss of Q A function and O 2 evolution. The aerobic photoinhibition also induced a decrease in the F m level of fluorescence, but its course was h much faster than the loss of O 2 evolution. Degradation of the D1 and D2 proteins followed the loss of O 2 evolution but it did not induce clear changes in whole structure of PS II as detected by FTIR even after prolonged photoinhibition. Based on these results, the functional and structural changes of PS II by the aerobic photoinhibition were discussed, in relation to the primary event of photoinhibition.

The 1-Hz fluorometer: A new approach to fast and sensitive long-term studies of active chlorophyll and environmental influences

A new instrument for environmental monitoring, called at 1-Hz fluorometer, provides two modes of application. First, it enables a quantitative determination of algal concentrations down to 20 ng/l. Second, it can be used as a biosensor for changes in environmental conditions. The distinction between the signals from living chlorophyll-containing algae and other fluorescent material is achieved by using two modulated light-sources resulting in a mean fluence rate of 200 μE. The measuring light induces changes in chlorophyll fluorescence (yield) with a frequency of 1 kHz, and the actinic light modulates the redox state of the quenchers of PS II with a frequency of 1 Hz. This leads to a modulation of the yield which is detected by two phase-sensitive rectifiers (double correlation). Measurements from different sites in a river, and in the Baltic and North Seas, show that correction by the built-in simultaneously-measured attenuation is necessary in order to obtain values which are identical with those determined by a photometric analysis (Uvikon 860). This applies if the transmission becomes less than about 95%. Suspensions ofDunaliella salina exposed to ammonia and phosphate were used for illustrating the usage for environmental monitoring. It is shown that this system can measure changes in the chlorophyll fluorescence of living algae caused by changes in concentration of ammonia down to 1 μg/l and of phosphate down to 10 μg/l.

In situ evidence that chilling in the light does not cause uncoupling of photophosphorylation or detachment of coupling factor in chilling-sensitive plants

The potential involvement of impaired photophosphorylation in the chilling sensitivity of photosynthesis in warm climate plant species has been a topic of investigation for more than two decades. With recent advances in the analysis of photosynthetic energy transduction in intact leaves, experiments are now possible that either address or avoid important uncertainties in the significance and interpretation of earlier in vitro work. Nevertheless, different laboratories using different techniques to analyze the effects of chilling in the light on photophosphorylation in intact cucumber (Cucumis sativus) leaves have come to very different conclusions regarding the role of impaired ATP formation capacity in the inhibition of net photosynthesis. In order to evaluate these discrepancies and bring this issue to a final resolution, in this investigation, we have made a detailed analysis of the decay of the flash-induced electrochromic shift and changes in chlorophyll fluorescence yield in cucumber leaves before, during and after a 5 h light-chill at chill temperatures of between 4 and 10°C. We feel that our findings address the major discrepancies in both data and interpretation as well as provide convincing evidence that photophosphorylation is not disrupted in cucumber leaves during or after light and chilling exposure. It follows that impaired photophosphorylation is not a contributing element to the inhibition of net photosynthesis that is widely observed in warm climate plants as a result of chilling in the light.

The Effects of Excess Irradiance on Photosynthesis in the Marine Diatom Phaeodactylum tricornutum.

The response of Phaeodactylum tricornutum to excess light was remarkably similar to that observed in higher plants and green algae and was characterized by complex changes in minimal fluorescence yields of fully dark-adapted samples and declines in maximum variable fluorescence levels and oxygen evolution rates. In our study the parallel decreases in the effective rate constant for photosystem II (PSII) photochemistry, the variable fluorescence yield of a dark-adapted sample, and light-limited O2 evolution rates after short (0-10 min) exposures to photoinhibitory conditions could not be attributed to damage or down-regulation of PSII reaction centers. Instead, these changes were consistent with the presence of nonphotochemical quenching of PSII excitation energy in the antennae. This quenching was analogous to that component of nonphotochemical quenching studied in higher plants that is associated with photoinhibition of photosynthesis and/or processes protecting against photoinhibition in that it did not relax readily in the dark and persisted in the absence of a bulk transthylakoid proton gradient. The quenching was most likely associated with photoprotective processes in the PSII antenna that reduced the extent of photoinhibitory damage, particularly after longer exposures. Our results suggest that a large population of damaged, slowly recovering PSII centers did not form in Phaeodactylum even after 60 min of exposure to excess actinic light.

SHORT‐TERM RESPONSE OF THE DIADINOXANTHIN CYCLE AND FLUORESCENCE YIELD TO HIGH IRRADIANCE IN CHAETOCEROS MUELLERI (BACILLARIOPHYCEAE)1

ABSTRACTThe relationship between the diadinoxanthin cycle and changes in fluorescence yield in the diatom Chaetoceros muelleri Lemm. (clone CH10, Amorient Aquafarm, Inc., Hawaii) was investigated. High‐light‐induced changes in fluorescence yield and xanthophyll de‐epoxidation occurred very rapidly (first order rate constant 1.60 min−1). The observed light‐induced changes in diatoxanthin and diadinoxanthin concentration were consistent with a two‐pool scheme for diadinoxanthin, one of which does not undergo de‐epoxidation. Changes in xanthophyll concentration correlated with changes in in vivo absorbance indicating that diadinoxanthin cycle activity in vivo can be monitored spectrophotometrically. However, changes in cell absorbance were small relative to total optical absorption cross section. Increases in the concentration of diatoxanthin were linearly correlated with increases in the rate constant for thermal de‐excitation in the antenna of photosystem II (PSII). Antenna quenching produced or mediated by diatoxanthin may, thus, protect the PSII reaction center in diatoms. Changes in the maximum fluorescence yield suggested that changes in the reaction center also contributed to nonphotochemical quenching of fluorescence. Thus, reaction center quenching affected the relationship between antenna quenching and changes in photochemical efficiency producing the effect of a decrease in fluorescence yield without a decrease in photochemical efficiency.

2-Aminopurine optical spectra: Solvent, pentose ring, and DNA helix melting dependence

2-Aminopurine (2AP) absorption and fluorescence excitation and emission spectra in a series of solvents have been measured to assess effects of solvent polarity. Emission spectra of the free base shift to the red in solvents of a higher dielectric constant, including water but excepting dioxane. Excitation spectra also red-shift, except in water. A change in dipole moment of 2.8 D upon excitation is obtained from a Bilot-Kawski plot which includes data from potentially anomolous solvents such as alcohols but which excludes dioxane and aqueous solvents. Attachment of ribose or 2'-deoxyribose causes 1 to 2-nm shifts in the position of fluorescence excitation and emission spectra of 2AP in water and little change in fluorescence yield. Melting of the DNA duplex d[CTGA(2AP)TTCAG]2 yields a blue shift of the excitation and no shift of the emission spectrum-results consistent with increased exposure to water and formation of 2AP-water H bonds in the ground state. The spectral shift occurs 5°C or more below the helix melting temperature, implying a premelting structural change in the decamer.

X-Ray Standing Wave Technique as a Tool in the Study of the Imperfect Crystals

The application of the X-ray standing wave (XSW) technique in the case of imperfect crystals meets serious theoretical and interpretational problems. However, well-known advantages of the XSW technique make it especially interesting to the study of the ordering of impurities and of various processes leading to changes in this ordering in various likes of imperfect crystals. In this work we try to answer the question how imperfection of a crystal may influence the changes in fluorescence yield during the XSW measurement. Two likes of imperfect crystals are studied: Si(111) implanted with high energetic Bi+ ions, and Ζn1-xCoxSe single crystal with natural (111) face. The discussion of obtained results shows that general features of the X-ray standing wave field are conserved despite the considerable imperfections of the crystals. The results seem to support the applicability of the XSW technique to the study of imperfect materials, although some further theoretical effort would be required. PACS numbers: 61.10.-i

Short‐term variations of the bio‐optical properties of the ocean in response to cloud‐induced irradiance fluctuations

Variations in natural irradiance caused by changing cloud cover were examined as a potential source for the variability of the bio‐optical properties in the open ocean. Time series of the scalar irradiance (PAR), beam attenuation coefficient (at 660 nm), stimulated chlorophyll fluorescence, concentration of dissolved oxygen, and water temperature were subject to spectral analysis. These data were acquired in the Sargasso Sea at a depth of 23 m during the Biowatt mooring experiment in 1987. Three cloudy days, on which power spectra of PAR exhibited distinct maxima at periods of about 40 to 100 min, were selected for the analysis. Similar variations occurred in beam attenuation, fluorescence and dissolved oxygen as evidenced by relatively high coherence (> 0.5) between PAR and these variables. This is suggested to be related to rapid photoadaptive responses in the phytoplankton community on time scales of minutes. Possible explanations for these responses include changes in fluorescence yield, photosynthetic rate, cellular absorption, cell size and refractive index. No relationship between PAR and the bio‐optical parameters was observed when hydrodynamical factors prevailed over time scales similar to those for cloud‐induced irradiance fluctuations. The hydrodynamical effects were successfully traced by water temperature variations which, in further studies, may prove useful for separation of phenomena controlling within‐day bio‐optical variability in the ocean.

Transient chlorophyll fluorescence yield changes from the isolated photosystem II reaction centre induced by electron transfer

It is shown that the photoaccumulation of reduced pheophytin can occur in the isolated D1/D2/cytochrome b559 photosystem II (PSII) reaction centre when Fe(II) is used as a donor to P680 + in a way similar to that observed in the presence of sodium dithionite. Using Fe(II) as an electron donor system it has been possible to induce chlorophyll fluorescence transients from the isolated reaction centre complex when it is reconstituted with decylplastoquinone. These transients seem to reflect classical changes in fluorescence yield of PSII which accompany the conversion of the quinone acceptor from its oxidised to its reduced state. In the case of the isolated complex these decyplastoquinone dependent changes in fluorescence yield are dominated by direct quenching by oxidised quinone and do not reflect changes in the extent of the recombination reaction between the primary radical pair, P680 + pheophytin _.

Effects of O2 and CO2 Concentrations on Quantum Yields of Photosystems I and II in Tobacco Leaf Tissue

The interactive effects of irradiance and O(2) and CO(2) levels on the quantum yields of photosystems I and II have been studied under steady-state conditions at 25 degrees C in leaf tissue of tobacco (Nicotiana tabacum). Assessment of radiant energy utilization in photosystem II was based on changes in chlorophyll fluorescence yield excited by a weak measuring beam of modulated red light. Independent estimates of photosystem I quantum yield were based on the light-dark in vivo absorbance change at 830 nanometers, the absorption band of P700(+). Normal (i.e. 20.5%, v/v) levels of O(2) generally enhanced photosystem II quantum yield relative to that measured under 1.6% O(2) as the irradiance approached saturation. Photorespiration is suspected to mediate such positive effects of O(2) through increases in the availability of CO(2) and recycling of orthophosphate. Conversely, at low intercellular CO(2) concentrations, 41.2% O(2) was associated with lower photosystem II quantum yield compared with that observed at 20.5% O(2). Inhibitory effects of 41.2% O(2) may occur in response to negative feedback on photosystem II arising from a build-up in the thylakoid proton gradient during electron transport to O(2). Covariation between quantum yields of photosystems I and II was not affected by concentrations of either O(2) or CO(2). The dependence of quantum yield of electron transport to CO(2) measured by gas exchange upon photosystem II quantum yield as determined by fluorescence was unaffected by CO(2) concentration.

Inhibition of the Chloroplast Photochemical Reactions by Treatment of Wheat Seedlings with Low Concentrations of Cadmium: Analysis of Electron Transport Activities and Changes in Fluorescence Yield

2+ ions for 24 h in dark, and after this treatment the plants were grown in the light until the primary leaves were fully developed. Cd 2+ ions (30-120 fiu) induced a significant decrease in activities of both photosystem II and photosystem I. The extent of the decrease in PS II activity was much greater than that in the PS I activity. Analysis of changes in the fluorescence yield of chlorophyll a also indicated that Cd 2+ ions drastically affect the photochemistry of photosystem II. Cd 2+ ions induced decrease in the rates of photoreduction of 2,6-dichlorophenol indophenol even in the presence of the exogenous electron donor, hydroxylamine, both in Tris-treated and untreated chloroplasts. This result suggests that the site of inhibition is near the site of donation of electrons by hydroxylamine. Treatment with Cd 2+ ions impairs the electron transport system on the reducing side of PS II. The decrease in the fluorescence yield of Chi a is less than that in the evolution of O2 mediated by oxidized phenylenediamine. This difference may be a result of inhibition on the reducing side of PS II. In addition to inhibition on the reducing side, Cd 2+ ions may affect the oxidizing side of PS II. A comparative study of the rates of evolution of O2 with /7-benzoquinone and dichloro-^7-benzoquinone as electron acceptors was performed since the halogenated benzoquinones have been shown to accept electrons from both active and inactive centers of photosystem II while some of the benzoquinones accept electrons only from active centers. The results suggest that Cd 2+ ions induced a marginal increase in the number of inactive reaction centers in PS II. Analysis of light-saturation-kinetics of the evolution of O2 catalysed by PS II indicates a reduction in the size of the antennae as well as in the concentration of the active (a-type) reaction centers of PS II. Thus, the Cd 2+ -induced effects on the photochemistry of PS II involve changes on the reducing side of PS II as well as possible changes in the sizes of the populations of active and inactive centers. Thus, short-term exposure to Cd 2+ ions during establishment of seedlings has a severely detrimental effect on photochemical activities in chloroplasts.

Dissipation of excitation energy by Photosystem II particles at low pH

The effect of decreasing the pH on the properties of Photosystem II particles isolated from pea and spinach has been investigated. Between pH 6.5 and 4.0 there was a decrease in quantum efficiency of oxygen evolution that was not associated with a proportional closure of reaction centres, as determined by the fluorescence coefficient, qP. This decrease in the quantum yield of Photosystem II appeared to be due to two separate mechanisms. Between pH 6.5 and 5.0, the decrease in quantum yield was not associated with any change in fluorescence yield or in the ratio of Fv/Fm; the decrease was greater at limiting light than in saturating light and may be caused by a cyclic electron transfer that is associated with the destabilisation of the oxidised intermediates of the oxygen-evolving complex and an accelerated rate of Q−A oxidation. Below pH 5.0 the decrease in efficiency is correlated with a quenching of chlorophyll fluorescence. This quenching was light-dependent and required oxidising conditions; redox titrations indicated that a component with a midpoint oxidation reduction potential of +405 mV at pH 4.0 needed to be oxidised if the low pH-dependent quenching was to be observed. The relationship between these effects of low pH and the ‘down-regulation’ of Photosystem II observed under physiological conditions is discussed.

A portable multi-flash kinetic fluorimeter for measurement of donor and acceptor reactions of Photosystem 2 in leaves of intact plants under field conditions

A newly-developed field-portable multi-flash kinetic fluorimeter for measuring the kinetics of the microsecond to millisecond reactions of the oxidizing and reducing sides of photosystem 2 in leaves of intact plants is described and demonstrated. The instrumental technique is a refinement of that employed in the 'double-flash' kinetic fluorimeter (Joliot 1974 Biochim Biophys Acta 357: 439-448) where a low-intensity short-duration light pulse is used to measure the fluorescence yield changes following saturating single-turnover light pulses. The present instrument uses a rapid series of short-duration (2 μs) pulses to resolve a complete microsecond to millisecond time-scale kinetic trace of fluorescence yield changes after each actinic flash. Differential optics, using a matrix of optical fibers, allow very high sensitivity (noise levels about 0.05% Fmax) thus eliminating the need for signal averaging, and greatly reducing the intensity of light required to make a measurement. Consequently, the measuring pulses have much less actinic effect and an entire multi-point trace (seven points) excites less than 1% of the reaction centers in a leaf. In addition, bu combining the actinic and measuring pulse light in the optical fiber network, the tail of the actinic flash can be compensated for, allowing measurements of events as rapidly as 20 μs after the actinic flash. This resolution makes practical the routine measurement of the microsecond turnover kinetics of the oxygen evolving complex in leaves of intact plants in the field. The instrument is demonstrated by observing flash number dependency and inhibitor sensitivity of the induction and decay kinetics of flash-induced fluorescence transients in leaves of intact plants. From these traces the period-two oscillations associated with the turnover of the two-electron gate and the period-four oscillations associated with the turnover of the oxygen evolving complex can be observed. Applications of the instrument to extending our knowledge of chloroplast function to the whole plant, the effects on plants of environmental stress, herbicides, etc, and possible applications to screening of mutants are discussed.

The relationship between non-photochemical quenching of chlorophyll fluorescence and the rate of photosystem 2 photochemistry in leaves

It has been suggested previously that non-photochemical quenching of chlorophyll fluorescence is associated with a decrease in the rate of photosystem 2 (PS 2) photochemistry. In this study analyses of fluorescence yield changes, induced by flashes in leaves exhibiting different amounts of non-photochemical quenching of fluorescence, are made to determine the effect of non-photochemical excitation energy quenching processes on the rate of PS 2 photochemistry. It is demonstrated that both the high-energy state and the more slowly relaxing components of non-photochemical quenching reduce the rate of PS 2 photochemistry. Flash dosage response curves for fluorescence yield show that non-photochemical quenching processes effectively decrease the relative effective absorption cross-section for PS 2 photochemistry. It is suggested that non-photochemical quenching processes exert an effect on the rate of PS 2 photochemistry by increasing the dissipation of excitation energy by non-radiative processes in the pigment matrices of PS 2, which consequently results in a decrease in the efficiency of delivery of excitation energy for PS 2 photochemistry.