Effects of solar UV stress on chlorophyll fluorescence kinetics of intertidal macroalgae from southern Spain: a case study in Gelidium species

Photoinhibition and recovery kinetics after short exposure to solar radiation following three different irradiance treatments of irradiances (PAR, PAR+UVA and PAR+UVA+UVB) was assessed in two intertidal species of the genus Gelidium, Gelidium sesquipedale and G. latifolium, collected from Tarifa (southern Spain) using in vivo chlorophyll fluorescence (PAM fluorometry). After 3 h UV radiation exposure, optimal quantum efficiency (Fv/Fm) in G. sesquipedale decreased between 25 and 35% relative to the control. Under PAR alone, values decreased to 60%. In G. latifolium, photoinhibition did not exceed 40%. Similar results were found for the effective quantum yield (ΔF/Fm′), however, no marked differences in relation to light treatments were seen. When plants were shaded for recovery from stress, only in G. latifolium a significant increase in photosynthesis was observed (between 80 and 100% of control). In contrast, photosynthesis of G. sesquipedale suffered a chronic photoinhibition or photodamage under the three light irradiances. Full solar radiation (PAR+UVA+UVB) affected also the electron transport rate in both species. Here, initial slopes of electron transport vs. irradiance curves decreased up to 60% of controls. Although the recovery kinetic under PAR+UVA+UVB conditions was delayed in G. latifolium, after 24 h recovery this species reached significantly higher than G. sesquipedale. PAR impaired electron trasport only in G. sesquipedale. Overall, both species are characterized by different capacity to tolerate enhanced solar radiation. G. latifolium is a sun adapted plant, well suited to intertidal light conditions, whereas G. sesquipedale, growing at shaded sites in the intertidal zone, is more vulnerable to enhanced UV radiation. This revised version was published online in June 2006 with corrections to the Cover Date.     

PAM fluorometer based on medium-frequency pulsed Xe-flash measuring light: A highly sensitive new tool in basic and applied photosynthesis research

A newly developed modulation fluorometer is described which employs repetitive 1 s Xe-flashes for excitation light. Similar to the standard PAM Chlorophyll Fluorometer, which uses 1 s LED pulses for measuring light, the integrated measuring light intensity is sufficiently low to monitor the dark-fluorescence level, F_o. The maximal fluorescence yield, F_m, can be determined with high selectivity upon application of a saturating light pulse. The Xe-PAM displays exceptionally high sensitivity, enabling quenching analysis at chlorophyll concentrations as low as 1 g/l, thus allowing to assess photosynthesis of phytoplankton in natural waters like lakes, rivers and oceans. Due to high flexibility in the choice of excitation and emission wavelengths, this system also provides the experimental basis for a thorough study of fluorescence and photosynthesis properties of various algae classes with differing antenna organisation. By appropriate modifications, the instrument may as well be used to measure with great sensitivity and selectivity other types of fluorescence (e.g. NADPH-fluorescence), as well as light-scattering and absorbance changes.     

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.     

The effects of elevated light on Photosystem II function: A thermoluminescence study

We have used the technique of thermoluminescence (TL) to investigate high-light-induced chlorophyll fluorescence quenching phenomena in barley leaves, and have shown it to be a powerful tool in such investigations. TL measurements were taken from wild-type and chlorina f2 barley leaves which had been dark-adapted or exposed to 20 min illumination of varying irradiance or given varying periods of recovery following strong irradiance. We have found strong evidence that there is a sustained trans-thylakoid pH in leaves following illumination, and that this pH gives rise to quenching of chlorophyll fluorescence which has previously been identified as a slowly-relaxing component of antenna-related protective energy dissipation; we have identified a state of the PS II reaction centre resulting from high light treatments which is apparently able to perform normal charge separation and electron transport but which is non-photochemically quenched, in that the application of a light pulse of high irradiance cannot cause the formation of a high fluorescent state; and we have provided evidence that a transient state of the PS II reaction centre is formed during recovery from such high light treatments, in which electron transport from Q_Ato Q_Bis apparently impaired.     

On the rates of cyclic electron transport around Photosystem II in the presence of donor side limitation

Photosystem II cyclic electron transport was investigated at low pH in spinach thylakoids and PS II preparations from the cyanobacteriumPhormidium laminosum. Variable fluorescence (F_v) quenching at a very low light intensity was examined as an indicator of cyclic electron flow. A progressive quenching of F_v was observed as the pH was lowered; however, this was shown to be mainly due to an inhibition of oxygen evolution. Cyclic electron flow in the uninhibited centres was estimated to occur at a rate comparable to or smaller than 1 mole O₂ mg Chl^–1 h^–1 in the pH range 5.0 to 7.8.The quantum yeeld of oxygen production is known to decrease at low pH and has been taken to indicate cyclic electron flow (Crofts and Horton (1991) Biochim Biophys Acta 1058: 187–193). However, a direct all-or-none inhibition of oxygen production at low pH has also been reported (Meyer et al. (1989) Biochim Biophys Acta 974: 36–43). We have analysed the effects of light intensity on the rates of oxygen evolution in order to calculate _U, the quantum yield of open and uninhibited centres. _U was found to be constant over a broad pH range, and by using ferricyanide and phenyl-p-benzoquinone as electron acceptors the maximum possible rate of cyclic electron transport was equivalent to no more than 1 mole O₂ mg Chl^–1 h^–1. The rate was no greater when the acceptor was adjusted to provide the most favourable conditions for cyclic flow.     

Photoadaptation in the Green Alga Spongiochloris Sp. A Three-Fluorometer Study

The dark-adapted cells of the green alga Spongiochloris sp. were exposed to "white light" of 1000 μmol(photon) m⁻² s⁻¹ for 2 h and then dark adapted for 1.5 h. Changes of photochemical activities during photoadaptation were followed by measurement of chlorophyll (Chl) fluorescence kinetics, 77 K emission spectra, photosynthetic oxygen evolution, and pigment composition. We observed a build-up of slowly-relaxing non-photochemical quenching which led to a decrease of the F_v/F_m parameter and the connectivity. In contrast to the depression of F_v/F_m (35 %) and the rise of non-photochemical quenching (∼ 1.6), we observed an increase in effective absorption cross-section (20 %), Hill reaction (30 %), photosynthetic oxygen evolution (80 %), and electron transport rate estimated from the Chl fluorescence analysis (80 %). We showed an inconsistency in the presently used interpretation schemes, and ascribe the discrepancy between the increase of effective absorption cross-section and the photosynthetic activities on one side and the effective non-photochemical quenching on the other side to the build-up of a quenching mechanism which dissipates energy in closed reaction centres. Such a type of quenching changes the ratio between thermal dissipation and fluorescence without any effect on photochemical yield. In this case the F_v/F_m ratio cannot be used as a measure of the maximum photochemical yield of PS2. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

Photosynthetic response of <Emphasis Type="Italic">Quercus ilex</Emphasis> L. plants grown on compost and exposed to increasing photon flux densities and elevated CO<Subscript>2</Subscript>

Quercus ilex plants grown on two different substrates, sand soil (C) and compost (CG), were exposed to photosynthetic photon flux densities (PPFD) at 390 and 800 μmol(CO₂) mol⁻¹ (C₃₉₀ and C₈₀₀). At C₈₀₀ both C and CG plants showed a significant increase of net photosynthetic rate (P _N) and electron transport rate (ETR) in response to PPFD increase as compared to C₃₉₀. In addition, at C₈₀₀ lower non-photochemical quenching (NPQ) values were observed. The differences between C₃₉₀ and C₈₀₀ were related to PPFD. The higher P _N and ETR and the lower dissipative processes found in CG plants at both CO₂ concentrations as compared to C plants suggest that substrate influences significantly photosynthetic response of Q. ilex plants. Moreover, short-term exposures at elevated CO₂ decreased nitrate photo-assimilation in leaves independently from substrate of growth.     

Measurement of chlorophyll fluorescence within leaves using a modified PAM Fluorometer with a fiber-optic microprobe

By using a fiber-optic microprobe in combination with a modified PAM Fluorometer, chlorophyll fluorescence yield was measured within leaves with spatial resolution of approximately 20 m. The new system employs a miniature photomultiplier for detection of the pulse-modulated fluorescence signal received by the 20 m fiber tip. The obtained signal/noise ratio qualifies for recordings of fluorescence induction kinetics (Kautsky effect), fluorescence quenching by the saturation pulse method and determination of quantum yield of energy conversion at Photosystem II at different sites within a leaf. Examples of the system performance and of practical applications are given. It is demonstrated that the fluorescence rise kinetics are distinctly faster when chloroplasts within the spongy mesophyll are illuminated as compared to palisade chloroplasts. Photoinhibition is shown to affect primarily the quantum yield of the palisade chloroplasts when excessive illumination is applied from the adaxial leaf side. The new system is envisaged to be used in combination with light measurements within leaves for an assessment of the specific contributions of different leaf regions to overall photosynthetic activity and for an integrative modelling of leaf photosynthesis.This paper is dedicated to Ulrich Heber on the occasion of his 65th birthday, with great respect for his outstanding achievements in photosynthesis research.     

A simple model relating photoinhibitory fluorescence quenching in chloroplasts to a population of altered Photosystem II reaction centers

A model is presented describing the relationship between chlorophyll fluorescence quenching and photoinhibition of Photosystem (PS) II-dependent electron transport in chloroplasts. The model is based on the hypothesis that excess light creates a population of inhibited PS II units in the thylakoids. Those units are supposed to posses photochemically inactive reaction centers which convert excitation energy to heat and thereby quench variable fluorescence. If predominant photoinhibition of PS II and cooperativity in energy transfer between inhibited and active units are presumed, a quasi-linear correlation between PS II activity and the ratio of variable to maximum fluorescence, F_VF_M, is obtained. However, the simulation does not result in an inherent linearity of the relationship between quantum yield of PS II and F_VF_M ratio. The model is used to fit experimental data on photoinhibited isolated chloroplasts. Results are discussed in view of current hypotheses of photoinhibition.Abbreviations F_M maximum total fluorescence - F₀ initial fluorescence - F_V maximum variable fluorescence - PS Photosystem - Q_A, Q_B primary and secondary electron acceptors of Photosystem II     

Chloroplast energization and oxidation of P700/plastocyanin in illuminated leaves at reduced levels of CO2 or oxygen

Chlorophyll fluorescence, light scattering, the electrochromic shift P₅₁₅ and levels of some photosynthetic intermediates were measured in illuminated leaves. Oxygen and CO₂ concentrations in the gas phase were varied in order to obtain information on control of Photosystem II activity under conditions such as produced by water stress, when stomatal closure restricts access of CO₂ to the photosynthetic apparatus. Light scattering and energy-dependent fluorescence quenching indicated a high level of chloroplast energization under high intensity illumination even when linear electron transport was curtailed in CO₂-free air or in 1% oxygen with 35 ll^-1 CO₂. Calculations of the phosphorylation potential based on measurements of phosphoglycerate, dihydroxyacetone phosphate and NADP revealed ratios of intrathylakoid to extrathylakoid proton concentrations, which were only somewhat higher in air containing 35 l l^-1 CO₂ than in CO₂-free air or 1% oxygen/35 l l^-1 CO₂. Anaerobic conditions prevented appreciable chloroplast energization. Acceptor-limitation of electron flow resulted in a high reduction level of the electron transport chain, which is characterized by decreased oxidation of P₇₀₀, not only under anaerobic conditions, but also in air, when CO₂ was absent, and in 1% oxygen, when the CO₂ concentration was reduced to 35 ll^-1. Efficient control of electron transport was indicated by the photoaccumulation of P₇₀₀ ⁺ at or close to the CO₂ compensation point in air. It is proposed to require the interplay between photorespiratory and photosynthetic electron flows, electron flow to oxygen and cyclic electron flow. The field-indicating electrochromic shift (P₅₁₅) measured as a rapid absorption decrease on switching the light off followed closely the extent of photoaccumulation of P₇₀₀ ⁺ in the light.Abbreviations F, F₀, F₀, F_M, F_M chlorophyll fluorescence levels - GA glyceraldehyde - P₅₁₅ field indicating rapid absorption change peaking at 522 nm - Q_A primary quinone acceptor in Photosystem II - Q_N non-photochemical quenching of chlorophyll fluorescence - Q_q photochemical quenching of chlorophyll fluorescence     

Identification of Photosynthetic Mutants of Arabidopsis by Automatic Screening for Altered Effective Quantum Yield of Photosystem 2

Quantification of chlorophyll (Chl) fluorescence is a versatile tool for analysing the photosynthetic performance of plants in a non-intrusive manner. A pulse-amplitude modulated fluorometer was combined with a CNC router for the automated measurement of the effective quantum yield of photosystem 2 (₂) of Arabidopsis thaliana plants. About 90 000 individual plants representing 7 500 lines derived from En-transposon and T-DNA mutagenised Arabidopsis populations were screened for mutants with altered ₂. Forty-eight recessive ₂ mutations were identified of which most exhibit also altered pigmentation and increased photosensitivity. For three ₂ mutants the corresponding mutated genes were identified that code all for chloroplast-located proteins. Comparison of the ₂ mutant screen with other screening methods based on the measurement of Chl fluorescence shows that the ₂ mutants identified are different to mutants identified by high Chl fluorescence. Some ₂ mutants, on the contrary, are common to mutants identified by screens based on non-photochemical quenching.     

Coupled cyclic electron transport in intact chloroplasts and leaves of C3 plants: Does it exist? if so,what is its function?

Transthylakoid proton transport based on Photosystem I-dependent cyclic electron transport has been demonstrated in isolated intact spinach chloroplasts already at very low photon flux densities when the acceptor side of Photosystem I (PS I) was largely closed. It was under strict redox control. In spinach leaves, high intensity flashes given every 50 s on top of far-red, but not on top of red background light decreased the activity of Photosystem II (PS II) in the absence of appreciable linear electron transport even when excitation of PS II by the background light was extremely weak. Downregulation of PS II was a consequence of cyclic electron transport as shown by differences in the redox state of P700 in the absence and the presence of CO₂ which drained electrons from the cyclic pathway eliminating control of PS II. In the presence of CO₂, cyclic electron transport comes into play only at higher photon flux densities. At H⁺/e=3 in linear electron transport, it does not appear to contribute much ATP for carbon reduction in C3 plants. Rather, its function is to control the activity of PS II. Control is necessary to prevent excessive reduction of the electron transport chain. This helps to protect the photosynthetic apparatus of leaves against photoinactivation under light stress.     

On the relationship between the quantum yield of Photosystem II electron transport,as determined by chlorophyll fluorescence and the quantum yield of CO2-dependent O2 evolution

We tested the two empirical models of the relationship between chlorophyll fluorescence and photosynthesis, previously published by Weis E and Berry JA 1987 (Biochim Biophys Acta 894: 198–208) and Genty B et al. 1989 (Biochim Biophys Acta 990: 87–92). These were applied to data from different species representing different states of light acclimation, to species with C₃ or C₄ photosynthesis, and to wild-type and a chlorophyll b-less chlorina mutant of barley. Photosynthesis measured as CO₂-saturated O₂ evolution and modulated fluorescence were simultaneously monitored over a range of photon flux densities. The quantum yields of O₂ evolution (ØO₂) were based on absorbed photons, and the fluorescence parameters for photochemical (q_p) and non-photochemical (q_N) quenching, as well as the ratio of variable fluorescence to maximum fluorescence during steady-state illumination (F'_v/F'_m), were determined. In accordance with the Weis and Berry model, most plants studied exhibited an approximately linear relationship between ØO₂/q_p (i.e., the yield of O₂ evolution by open Photosystem II reaction centres) and q_N, except for wild-type barley that showed a non-linear relationship. In contrast to the linear relationship reported by Genty et al. for q_p×F'_v/F'_m (i.e., the quantum yield of Photosystem II electron transport) and ØCO₂, we found a non-linear relationship between q_p×F'_v/F'_m and ØO₂ for all plants, except for the chlorina mutant of barley, which showed a largely linear relationship. The curvilinearity of wild-type barley deviated somewhat from that of other species tested. The non-linear part of the relationship was confined to low, limiting photon flux densities, whereas at higher light levels the relationship was linear. Photoinhibition did not change the overall shape of the relationship between q_p×F'_v/F'_m and ØO₂ except that the maximum values of the quantum yields of Photosystem II electron transport and photosynthetic O₂ evolution decreased in proportion to the degree of photoinhibition. This implies that the quantum yield of Photosystem II electron transport under high light conditions may be similar for photoinhibited and non-inhibited plants. Based on our experimental results and theoretical analyses of photochemical and non-photochemical fluoresce quenching processes, we conclude that both models, although not universal for all plants, provide useful means for the prediction of photosynthesis from fluorescence parameters. However, we also discuss that conditions which alter one or more of the rate constants that determine the various fluorescence parameters, as well as differential light penetration in assays for oxygen evolution and fluorescence emission, may have direct effect on the relationships of the two models.Abbreviations F₀ and F'₀ fluorescence when all Photosystem II reaction centres are open in dark- and light-acclimated leaves, respectively - F_m and F'_m fluorescence when all Photosystem II reaction centres are closed in dark and light, respectively - F_v variable fluorescence equal to F_m-F₀ - F_s steady state level of fluorescence in light - F'_v and F'_m variable (F'_m-F'₀) and maximum fluorescence under steady state light conditions - HEPES N-2-hydroxyethylpiperazine-N-2-ethane-sulphonic acid - Q_A the primary, stabile quinone acceptor of Photosystem II - q_N non-photochemical quenching of fluorescence - q_p photochemical quenching of fluorescence - ØO₂ quantum yield of CO₂-saturated O₂ evolution based on absorbed photons     

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.     

Characterization of the Quenching of Chlorophyll a Fluorescence by β-Carotene Using the Non-Linear Analysis

Carotenoids (Car) regulate energy flow in photosynthesis by a specific Car-chlorophyll (Chl) interaction in the singlet-excited states, leading to a reduction in Chl fluorescence. We studied quenching of Chl a-fluorescence in benzene by trans--carotene. Non-linear analysis of the quenching process enables to explain the possible molecular mechanism leading to the de-excitation of Chl a. The fluorescence intensity was measured at 670 nm for excitation wavelengths of 380, 430, 640, and 650 nm. The -carotene concentrations ranged from 4×10^–5 M to 5×10^–3 M. When the samples were excited at 640 and 650 nm, the Stern-Volmer plots showed that the quenching process has high rate constants, hence -carotene is a very efficient quencher. Two different types of quenching process could take place.