Wavelength-dependent photodamage to <Emphasis Type="Italic">Chlorella</Emphasis> investigated with a new type of multi-color PAM chlorophyll fluorometer

A new type of multi-color PAM chlorophyll fluorometer (Schreiber et al. 2012) was applied for measurements of photodamage to photosystem II (PS II) in optically thin suspensions of Chlorella (200 μg Chl l^?1) in the presence of 1 mM lincomycin. An action spectrum of the relative decrease of F _v/F _m in the 440–625 nm range was measured, which not only showed the expected high activity in the blue, but at a lower level also substantial activity above 540 nm. With the same dilute suspension, a PS II absorption spectrum was derived via measurements of the O-I₁ rise kinetics induced by differently colored strong light at defined incident quantum flux densities. After normalization of the two spectra at 625 nm, the relative extent of photodamage at 440–480 nm proved substantially higher than absorption by PS II, whereas the two spectra were close to identical in the 540–625 nm range. Hence, overall photodamage to PS II appears to consist of two components, one of which is due to light absorbed by PS II pigments, whereas the other one is likely to involve direct light absorption by Mn in the oxygen-evolving complex (Hakala et al. Biochim Biophys Acta 1706:68–80, 2005). Based on this rationale, an action spectrum of the Mn mechanism of photodamage was deconvoluted from the overall action spectrum, declining steeply above 480 nm. An almost identical Mn-spectrum was derived by another approach with the PAR of the various colors being adjusted to give identical rates of PS II turnover, PAR _II. The tentative, basic assumption of negligibly small contribution of the Mn mechanism to photodamage above 540 nm was supported by supplementary measurements using an external 665 nm lamp. 665 nm not only gave about two times PS II turnover as compared to 625 nm, but also about two times photodamage.     

New type of dual-channel PAM chlorophyll fluorometer for highly sensitive water toxicity biotests

A new type of dual-channel PAM chlorophyll fluorometer has been developed, which is specialised in the detection of extremely small differences in photosynthetic activity in algae or thylakoids suspensions. In conjunction with standardised algae cultures or isolated thylakoids, the new device provides an ultrasensitive biotest system for detection of toxic substances in water samples. In this report, major features of the new device are outlined and examples of its performance are presented using suspensions of Phaeodactylum tricornutum (diatoms) and of freeze-dried thylakoids of Lactuca sativa (salad). Investigated and reference samples are exposed to the same actinic intensity of pulse-modulated measuring light. The quantum yields are assessed by the saturation pulse method. Clock-triggered repetitive measurements of quantum yield typically display a standard deviation of 0.1%, corresponding to the inhibition induced by 0.02 μg diuron l⁻¹. Hence, for diuron or compounds with similar toxicity, the detection limit is well below the 0.1 μg l⁻¹ defined as the limit for the presence of a single toxic substance in water by the European Commission drinking water regulation. The amounts of water and biotest material required for analysis are very small, as a single assay involves two 1 ml samples, each containing ca. 0.5 μg chlorophyll. Both with Phaeodactylum and thylakoids the relationship between inhibition and diuron concentration is strictly linear up to 10% inhibition, with very similar slopes. Apparent inhibition depends on the actinic effect of the measuring light, showing optima at 6 and 4 μmol quanta m⁻² s⁻¹ with Phaeodactylum and thylakoids, respectively. This revised version was published online in June 2006 with corrections to the Cover Date.     

利用调制荧光仪在线监测叶绿素荧光

介绍了利用便携式调制荧光仪PAM-2100在线监测叶绿素荧光的技术。该技术不影响植物的自然光合状态,司以在线监测Ft、F′m、Y、rETR、qP、qN或NPQ、PAR和叶温等指标。以凤眼莲为例进行了在线监测,每隔5min监测一次,共进行了225min的监测。结果表明叶绿索荧光参数的变化依赖于PAR的变化。Ft、rETR、qN和NPQ的变化与PAR的变化趋势一致,F′m、Y和qP的变化与PAR的变化相反。通过对风眼莲的在线监测,说明该技术是可靠的,具有简单、快速、灵敏等特点。随着新型调制荧光仪的出现,该技术可能在植物生态学领域得到广泛应用。     

Detection of rapid induction kinetics with a new type of high-frequency modulated chlorophyll fluorometer

A newly developed modulation fluorometer is described which operates with 1 sec light pulses from a light-emitting diode (LED) at 100 KHz. Special amplification circuits assure a highly selective recording of pulse fluorescence signals against a vast background of non-modulated light. The system tolerates ratios of up to 1:10⁷ between measuring light and actinic light. Thus it is possible to measure the dark fluorescence yield and record the kinetics of light-induced changes. A high time resolution allows the recording of the rapid relaxation kinetic following a saturating single turnover flash. Examples of system performance are given. It is shown that following a flash the reoxidation kinetics of photosystem II acceptors are slowed down not only by the inhibitor DCMU, but by a number of other treatments as well. From a light intensity dependency of the induction kinetics the existence of two saturated intermediate levels (I₁ and I₂) is apparent, which indicates the removal of three distinct types of fluorescence quenching in the overall fluorescence rise from F₀ to F_max.Abbreviations Q_A and Q_B consecutive electron acceptors of photosystem II - PS II photosystem II - P 680 reaction center chlorophyll of photosystem II - F₀ minimum fluorescence yield following dark adaptation - F_max maximum fluorescence yield - DCMU 3-(3, 4-dichlorophenyl)-1, 1-dimethyl-urea - DCCD N,N-dicyclohexylcarbodiimide - PQ plastoquinone - DAD diaminodurene Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement.     

Continuous recording of photochemical and non-photochemical chlorophyll fluorescence quenching with a new type of modulation fluorometer

A newly developed fluorescence measuring system is employed for the recording of chlorophyll fluorescence induction kinetics (Kautsky-effect) and for the continuous determination of the photochemical and non-photochemical components of fluorescence quenching. The measuring system, which is based on a pulse modulation principle, selectively monitors the fluorescence yield of a weak measuring beam and is not affected even by extremely high intensities of actinic light. By repetitive application of short light pulses of saturating intensity, the fluorescence yield at complete suppression of photochemical quenching is repetitively recorded, allowing the determination of continuous plots of photochemical quenching and non-photochemical quenching. Such plots are compared with the time courses of variable fluorescence at different intensities of actinic illumination. The differences between the observed kinetics are discussed. It is shown that the modulation fluorometer, in combination with the application of saturating light pulses, provides essential information beyond that obtained with conventional chlorophyll fluorometers.     

Estimating photosynthetic electron transport via chlorophyll fluorometry without Photosystem II light saturation

Estimates of thylakoid electron transport rates (J_e) from chlorophyll fluorometry are often used in combination with leaf gas exchange measurements to provide detailed information about photosynthetic activity of leaves in situ. Estimating J_e requires accurate determination of the quantum efficiency of Photosystem II (Φ_P), which in turn requires momentary light saturation of the Photosystem II light harvesting complex to induce the maximum fluorescence signal (F_M′). In practice, full saturation is often difficult to achieve, especially when incident photosynthetic photon flux density (Q) is high and energy is effectively dissipated by non-photochemical quenching. In the present work, a method for estimating the true F_M′ under high Q was developed, using multiple light pulses of varying intensity (Q′). The form of the expected relationship between the apparent F_M′ and Q′ was derived from theoretical considerations. This allowed the true F_M′ at infinite Q′ to be estimated from linear regression. Using a commercially available leaf gas exchange/ chlorophyll fluorescence measurement system, J_e was compared to gross photosynthetic CO₂ assimilation (A_G) under conditions where the relationship between J_e and A_G was expected to be linear. Both in C₄ leaves (Zea mays) in ambient air and also in C₃ leaves (Gossypium hirsutum) under non-photorespiratory conditions the apparent ratio between J_e and A_G declined at high Q when Φ_P was calculated from F_M′ measured simply using the highest available saturating pulse intensity. When F_M′ was determined using the multiple pulse / linear regression technique, the expected relationship between J_e and A_G at high Q was restored, indicating that the Φ_P estimate was improved. This method of determining F_M′ should prove useful for verifying when saturating pulse intensities are sufficient, and for accurately determining Φ_P when they are not. This revised version was published online in June 2006 with corrections to the Cover Date.     

How to correctly determine the different chlorophyll fluorescence parameters and the chlorophyll fluorescence decrease ratio R<Subscript>Fd</Subscript> of leaves with the PAM fluorometer

This contribution is a practical guide to the measurement of the different chlorophyll (Chl) fluorescence parameters and gives examples of their development under high-irradiance stress. From the Chl fluorescence induction kinetics upon irradiation of dark-adapted leaves, measured with the PAM fluorometer, various Chl fluorescence parameters, ratios, and quenching coefficients can be determined, which provide information on the functionality of the photosystem 2 (PS2) and the photosynthetic apparatus. These are the parameters F_v, F_m, F₀, F_m′, F_v′, NF, and ΔF, the Chl fluorescence ratios F_v/F_m, F_v/F₀, ΔF/F_m′, as well as the photochemical (q_P) and non-photochemical quenching coefficients (q_N, q_CN, and NPQ). q_N consists of three components (q_N = q_E + q_T + q_I), the contribution of which can be determined via Chl fluorescence relaxation kinetics measured in the dark period after the induction kinetics. The above Chl fluorescence parameters and ratios, many of which are measured in the dark-adapted state of leaves, primarily provide information on the functionality of PS2. In fully developed green and dark-green leaves these Chl fluorescence parameters, measured at the upper adaxial leaf side, only reflect the Chl fluorescence of a small portion of the leaf chloroplasts of the green palisade parenchyma cells at the upper outer leaf half. Thus, PAM fluorometer measurements have to be performed at both leaf sides to obtain information on all chloroplasts of the whole leaf. Combined high irradiance (HI) and heat stress, applied at the upper leaf side, strongly reduced the quantum yield of the photochemical energy conversion at the upper leaf half to nearly zero, whereas the Chl fluorescence signals measured at the lower leaf side were not or only little affected. During this HL-stress treatment, q_N, q_CN, and NPQ increased in both leaf sides, but to a much higher extent at the lower compared to the upper leaf side. q_N was the best indicator for non-photochemical quenching even during a stronger HL-stress, whereas q_CN and NPQ decreased with progressive stress even though non-photochemical quenching still continued. It is strongly recommended to determine, in addition to the classical fluorescence parameters, via the PAM fluorometer also the Chl fluorescence decrease ratio R_Fd (F_d/F_s), which, when measured at saturation irradiance is directly correlated to the net CO₂ assimilation rate (_{P N}) of leaves. This R_Fd-ratio can be determined from the Chl fluorescence induction kinetics measured with the PAM fluorometer using continuous saturating light (cSL) during 4–5 min. As the R_Fd-values are fast measurable indicators correlating with the photosynthetic activity of whole leaves, they should always be determined via the PAM fluorometer parallel to the other Chl fluorescence coefficients and ratios.     

Regulation of photosynthetic electron transport

The photosynthetic electron transport chain consists of photosystem II, the cytochrome b(6)f complex, photosystem I, and the free electron carriers plastoquinone and plastocyanin. Light-driven charge separation events occur at the level of photosystem II and photosystem I, which are associated at one end of the chain with the oxidation of water followed by electron flow along the electron transport chain and concomitant pumping of protons into the thylakoid lumen, which is used by the ATP synthase to generate ATP. At the other end of the chain reducing power is generated, which together with ATP is used for CO(2) assimilation. A remarkable feature of the photosynthetic apparatus is its ability to adapt to changes in environmental conditions by sensing light quality and quantity, CO(2) levels, temperature, and nutrient availability. These acclimation responses involve a complex signaling network in the chloroplasts comprising the thylakoid protein kinases Stt7/STN7 and Stl1/STN7 and the phosphatase PPH1/TAP38, which play important roles in state transitions and in the regulation of electron flow as well as in thylakoid membrane folding. The activity of some of these enzymes is closely connected to the redox state of the plastoquinone pool, and they appear to be involved both in short-term and long-term acclimation. This article is part of a Special Issue entitled "Regulation of Electron Transport in Chloroplasts".     

Inhibition of photosynthetic electron transport and formation of inactive chlorophyll in winter stressed Pinus silvestris

Kinetics of fluorescence at room temperature, electron transport and photooxidation of P700 and cytochrome f have been studied in chloroplasts isolated from active and winter stressed Pinus silvestris. The winter stress induced block in the electron transport chain between the two photosystems is close to the site of plastoquinone, since winter stress and DCMU caused the same type of inhibition of the reoxidation of the primary electron acceptor Q of photosystem II. No winter inhibition of the electron transport between cytochrome f and P700 was observed. Time course studies of P700 photooxidation in chloroplasts of active and winter stressed pine have shown that the photosynthetic unit size must be about equal in the two types of chloroplasts. An apparent increase of the photosynthetic unit size was induced by winter stress, as revealed by the high chlorophyll/P700 ratio of winter stressed pine. The phenomenon is explained by the formation of photosynthetically inactive chlorophyll. Low-temperature fluorescence emission spectra were recorded when either chlorophyll a (433 nm) or chlorophyll b (477 nm) were preferentially excited. Winter stress induced the formation of a chlorophyll a fraction emitting at 673 nm. This chlorophyll is most likely derived from the chlorophyll a antennae of the two photosystems, and it probably contributes to the photosynthetically inactive pool of chlorophyll in winter stressed pine. The light harvesting chlorophyll a/b complex is relatively resistant to winter stress.     

Evaluation of instant light‐response curves of chlorophyll fluorescence parameters obtained with a portable chlorophyll fluorometer on site in the field

Miniaturized pulse‐amplitude modulated photosynthesis yield analysers are primarily designed for measuring effective quantum yield (ΔF/F_m′) of photosystem II under momentary ambient light conditions in the field. Although this provides important ecophysiological information, it is often necessary to learn more about the potential intrinsic capacities of leaves by measuring light‐response curves. Thus, instruments provide light‐curve programmes, where light intensities are increased in short intervals and instant light‐response curves are recorded within a few minutes. This method can be criticized because photosynthesis will most likely not be in steady state. This technical report shows that with the appropriate precautions instant light curves can nevertheless provide reliable information about cardinal points of photosynthesis. First, the geometry of the light source of the instrument in relation to the quantum sensor must be considered and quantum sensor readings must be corrected. Second, the measurements of the light‐response curves must be compared with readings of effective quantum yield of photosystem II under ambient light conditions where photosynthesis is in steady state. This may show that in the critical range of the light curves either both measurements perfectly coincide or are offset against each other by a constant value (examples are given here). In the first case results of light curves can be taken at face values, and in the second case a simple correction can be applied. With these precautions and careful interpretations instant light‐response curves can be an enormous advantage in ecophysiological field work.     

Probing of photosynthetic reactions in four phytoplanktonic algae with a PEA fluorometer

High-resolution light-induced kinetics of chlorophyll fluorescence (OJIP transients) were recorded and analyzed in cultures of diatoms (Thalassiosira weissflogii, Chaetoceros mulleri) and dinoflagellates (Amphidinium carterae, Prorocentrum minimum). Fluorescence transients showed the rapid exponential initial rise from the point O indicating low connectivity between PS II units and high absorption cross-section of PS II antenna. Dark-adapted dinoflagellates revealed capability to maintain the PS I-mediated re-oxidation of the PQ pool at the exposure to strong actinic light that may lead to the underestimation of F _M value. In OJIP transients recorded in phytoplanktonic algae the fluorescence yield at the point O exceeded F _O level because Q_A has been already partly reduced at 50 μs after the illumination onset. PEA was also employed to study the recovery of photosynthetic reactions in T. weissflogii during incubation of nitrogen starved cells in N-replete medium. N limitation caused the impairment of electron transport between Q_A and PQs, accumulation of closed PS II centers, and the reduced ability to generate transmembrane ΔpH upon illumination, almost fully restored during the recovery period. The recovered cells showed much higher values of NPQ than control ones suggesting maximization of photoprotection mechanisms in the population with a ‘stress history.’     

Chromatic photoacclimation, photosynthetic electron transport and oxygen evolution in the chlorophyll d-containing oxyphotobacterium Acaryochloris marina

Changes in photosynthetic pigment ratios showed that the Chlorophyll d-dominated oxyphotobacterium Acaryochloris marina was able to photoacclimate to different light regimes. Chl d per cell were higher in cultures grown under low irradiance and red or green light compared to those found when grown under high white light, but phycocyanin/Chl d and carotenoid/Chl d indices under the corresponding conditions were lower. Chl a, considered an accessory pigment in this organism, decreased respective to Chl d in low irradiance and low intensity non-white light sources. Blue diode PAM (Pulse Amplitude Modulation) fluorometry was able to be used to measure photosynthesis in Acaryochloris. Light response curves for Acaryochloris were created using both PAM and O(2) electrode. A linear relationship was found between electron transport rate (ETR), measured using a PAM fluorometer, and oxygen evolution (net and gross photosynthesis). Gross photosynthesis and ETR were directly proportional to one another. The optimum light for white light (quartz halogen) was about 206+/-51 micromol m(-2) s(-1) (PAR) (Photosynthetically Active Radiation), whereas for red light (red diodes) the optimum light was lower (109+/-27 micromol m(-2) s(-1) (PAR)). The maximum mean gross photosynthetic rate of Acaryochloris was 73+/-7 micromol mg Chl d(-1) h(-1). The gross photosynthesis/respiration ratio (P(g)/R) of Acaryochloris under optimum conditions was about 4.02+/-1.69. The implications of our findings will be discussed in relation to how photosynthesis is regulated in Acaryochloris.     

Effect of phloridzin on photosynthetic electron transport

Phloridzin (2',4',6',4-tetraoxyhydrochalcon-2'-glucoside) was used to study the localization of synthesis of ATP in the electrontransporting chain of photosynthesis. It was shown that phloridzin inhibits the rate of photoreduction of NADP+ by isolated pea chloroplasts by 40%, electron transport via cytochrome f by 100% and via plastocyanin--by 50%. The "crossover" experiments demonstrated that phloridzin inhibits ADP-induced photoreduction of cytochrome f, having no effect on plastocyanin under identical conditions. It is assumed that the site of ATP synthesis is localized on the reduced site of cytochrome f, while the carrier itself is located in the electron transporting chain coupled to phosphorylation. It is possible that only part of the plastocyanin molecules are located in the phosphorylating pathway of electron transport.     

Regulation of the photosynthetic electron transport chain

The regulation of electron transport between photosystems II and I was investigated in the plant Silene dioica L. by means of measurement of the kinetics of reduction of P₇₀₀ following a light-to-dark transition. It was found that, in this species, the rate constant for P₇₀₀ reduction is sensitive to light intensity and to the availability of CO₂. The results indicated that at 25 °C the rate of electron transport is down-regulated by approximately 40–50% relative to the maximum rate achievable in saturating CO₂ and that this down-regulation can be explained by regulation of the electron transport chain itself. Measurements of the temperature sensitivity of this rate constant indicated that there is a switch in the rate-limiting step that controls electron transport at around 20 °C: at higher temperatures, CO₂ availability is limiting; at lower temperatures some other process regulates electron transport, possibly a diffusion step within the electron transport chain itself. Regulation of electron transport also occurred in response to drought stress and sucrose feeding. Measurements of non-photochemical quenching of chlorophyll fluorescence did not support the idea that electron transport is regulated by the pH gradient across the thylakoid membrane, and the possibility is discussed that the redox potential of a stromal component may regulate electron transport. Received: 4 March 1999 / Accepted: 25 May 1999     

The pigment stoichiometry in a chlorophyll a/c type photosynthetic antenna

The trimeric fucoxanthin-chlorophyll a/c protein (FCP) was purified from a Japanese brown alga, Cladosiphon okamuranus TOKIDA. Its pigment stoichiometry was determined to be chlorophyll (Chl) a:Chl c (1):Chl c (2):fucoxanthin?=?4.6:1.1:1.0:5.5 by a combination of binary HPLC and (1)H NMR spectroscopy. No violaxanthin found bound to the FCP. The ratio of Chl c/Chl a in this FCP is amongst the highest so far reported.     

Reprint of: Regulation of photosynthetic electron transport

The photosynthetic electron transport chain consists of photosystem II, the cytochrome b(6)f complex, photosystem I, and the free electron carriers plastoquinone and plastocyanin. Light-driven charge separation events occur at the level of photosystem II and photosystem I, which are associated at one end of the chain with the oxidation of water followed by electron flow along the electron transport chain and concomitant pumping of protons into the thylakoid lumen, which is used by the ATP synthase to generate ATP. At the other end of the chain reducing power is generated, which together with ATP is used for CO(2) assimilation. A remarkable feature of the photosynthetic apparatus is its ability to adapt to changes in environmental conditions by sensing light quality and quantity, CO(2) levels, temperature, and nutrient availability. These acclimation responses involve a complex signaling network in the chloroplasts comprising the thylakoid protein kinases Stt7/STN7 and Stl1/STN7 and the phosphatase PPH1/TAP38, which play important roles in state transitions and in the regulation of electron flow as well as in thylakoid membrane folding. The activity of some of these enzymes is closely connected to the redox state of the plastoquinone pool, and they appear to be involved both in short-term and long-term acclimation. This article is part of a Special Issue entitled: Regulation of Electron Transport in Chloroplasts.     

A new evaluation of chlorophyll absorption in photosynthetic membranes

The three major chlorophyll-proteins of spinach chloroplasts were solubilized with digitonin and isolated by electrophoresis with deoxycholate. The gel bands were identified from their absorption and fluorescence spectra measured at 77 K. The slowest moving band was a Photosystem I complex (CPI); the second, a Photosystem II complex (Cpa); and the third, a chlorophyll a-b, antenna complex (LHCP). When absorption spectra (630–730 nm) of the bands were added in the proportions found in the gel, the sum closely matched the absorption of the chloroplasts both before and after solubilization. Thus these spectra represent the native absorption of the major antenna chlorophyll-proteins of green plants. Each of these spectra was resolved with a computer assisted, curve-fitting program into 8 mixed Gaussian-Lorentzian shaped components. The major, Chl a components in the 3 fractions were different both in peak positions and bandwidths. This result suggests that each chlorophyll-protein has its own unique set of chlorophyll a spectral forms or components.Abbreviations Chl chlorophyll - CPI Photosystem I Chl-protein - CPa Photosystem II Chl-protein - LHCP light-harvesting Chl a-b protein - DOC sodium deoxycholate - SDS sodium dodecylsulfate CIW-DPB No. 819