Fast Fluorescence Quenching from Isolated Guard Cell Chloroplasts of Vicia faba Is Induced by Blue Light and Not by Red Light

Chlorophyll a fluorescence transients from isolated Vicia faba guard cell chloroplasts were used to probe the response of these organelles to light quality. Guard cell chloroplasts were isolated from protoplasts by passing them through a 10-μm nylon net. Intact chloroplasts were purified on a Percoll gradient. Chlorophyll a fluorescence transients induced by actinic red or blue light were measured with a fluorometer equipped with a measuring beam. Actinic red light induced a monophasic quenching, and transients induced by blue light showed biphasic kinetics having a slow and a fast component. The difference between the red and blue light-induced transients could be observed over a range of fluence rates tested (200-800 μmol m⁻² s⁻¹). The threshold fluence rate of blue light for the induction of the fast component of quenching was 200 μmol m⁻² s⁻¹, but in the presence of saturating red light, fluence rates as low as 25 μmol m⁻² s⁻¹ induced the fast quenching. These results indicate that guard cell chloroplasts have a specific response to blue light.     

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.     

The Effect of Photoinhibition on Photosynthetic Oxygen Production in the Brown Alga Dictyota dichotoma

Photoinhibition of photosynthesis in the brown alga, Dictyota dichotoma, was studied with a PAM fluorometer (Walz, Effeltrich, Germany) and a homemade oxygen measuring device. As a measure of fluorescence, Fv/Fm, and for the photosynthetic yield, ΔF/Fm', were used. Oxygen measurements show clearly that the observed degree, as well as the time course, of photoinhibition depends on the fluence rate of the light used to measure changes of the production rate. After photoinhibition of photosynthesis the depression of oxygen production caused by non-saturating fluence rates was generally much more pronounced than that caused by saturating or nearly saturating fluence rates. At minimal photoinhibition the initial slope and the convexity of the fluence rate-response curve of oxygen evolution decrease, whereas the level of light saturation decreases only after strong photoinhibition. Nevertheless, at different degrees of photoinhibition, changes in the degree of the upper bending of the fluence rate-response curve of oxygen production are also linearly correlated to changes in the fluorescence ratios (Fv/Fm and ΔF/Fm'). The action spectrum of photoinhibition, calculated on the basis of changes of Fv/Fm, indicates that the reaction center of PS I is not involved in photoinhibition. The lower effectiveness of blue light in comparison to effects of green and red light may be due to chloroplast displacement, as in the so-called strong light position, the light absorbed by the thalli in vivo is decreased.     

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.     

Salts and chloroplast fluorescence

Chloroplast fluorescence was excited by a weak measuring beam. A time-separated actinic light was used to modify the redox states of Q which in turn induced a change in the fluorescence yield. In salt-depleted chloroplasts, fluorescence saturated at a low actinic light intensity. CaCl₂ increased the “variable” fluorescence as well as the rate of ferricyanide-Hill reaction. With Tris-washed chloroplasts, Photosystem II donor couple, phenylenediamine and ascorbate, did not increase the fluorescence to a large extent without the presence of CaCl₂. It is suggested that salt-depletion inactivates the Photosystem II reaction center of chloroplasts.     

Effect of light intensity on chlorophyll fluorescence in wheat leaves: Application of PAM-fluorometry

Application of pulse-amplitude-modulation (PAM) fluorometers for measuring slow stages of chlorophyll fluorescence induction (CFI) is considered. With an example of Triticum aestuvum L. plants grown under continuous illumination at a photon flux density of 600 μmol/(m² s) photosynthetically active radiation (PAR), the CFI curves were analyzed with leaves of various ages as a function of actinic light intensity. The fluorometer PAM-2100 was applied for measurements of CFI curves. The characteristic peaks of CFI curves in wheat leaves were most conspicuous and had the largest amplitudes at 600–800 μmol/(m² s) PAR, which corresponds to the middle range of actinic light intensities employed in PAM-2100 fluorometers. In plants exposed to favorable and stressful conditions, the developmental stages may proceed at different rates; thus, the comparison of fluorescence parameters for leaves of equal calendar age but having different physiological states may provide ambiguous data. Therefore, the feasibility of recording CFI curves of different types is quite important for rapid diagnostics of the age and state of plant leaves, as well as for adequate physiological conclusions.     

Adaptation of a PAM-fluorometer for remote sensing of chlorophyll fluorescence

The Laser-PAM described in this paper is an adaptation of the PAM 101 fluorometer (Heinz Walz, Effeltrich, Germany) designed for remote sensing and non-invasive laboratory measurements of chlorophyll fluorescence. It is based on a 5 mW laser diode, emitting at 638 nm, and a Fresnel lens coupled to the ED-101 detection unit. Due to these modifications, measurements can be performed at a distance ranging from 0.3 to 2 m. The ED-101 detection unit has been modified to perform simultaneous measurements of both modulated fluorescence and light reflected by the leaf. Reflected light showed a good estimation of the photosynthetically active radiation measured exactly at the same area as the fluorescence. A particular advantage of the Laser-PAM fluorometer is its suitability for remote measurements under field conditions. Simultaneous fluorescence and gas-exchange measurements, performed on grapevine leaves, are reported as an example of an application for the Laser-PAM. This revised version was published online in June 2006 with corrections to the Cover Date.     

Substrate uptake mechanisms for yeast cells

Summary This paper reports on experiments in which a fluorometer, which permits culture fluorescence to be measured in situ was coupled to a bioreactor. The fluorometer through external light excitement NADH can be made to fluoresce, thus allowing intracellular NADH profiles to be measured in response to extracellular step-changes in substrate concentration. Experiments with baker's yeast and Candida tropicalis as organisms and glucose and hexadecane as substrates are reported. The results suggest different uptake mechanisms depending on cell type and on substrate.     

Measurement of gradients of absorbed light in spinach leaves from chlorophyll fluorescence profiles

Profiles of chlorophyll fluorescence were measured in spinach leaves irradiated with monochromatic light. The characteristics of the profiles within the mesophyll were determined by the optical properties of the leaf tissue and the spectral quality of the actinic light. When leaves were infiltrated with 10^?4M DCMU [3‐(3,4‐dichlorophenyl)‐1, 1‐dimethyl‐urea] or water, treatments that minimized light scattering, irradiation with 2000 μmol m^?2 s^?1 green light produced broad Gaussian‐shaped fluorescence profiles that spanned most of the mesophyll. Profiles for chlorophyll fluorescence in the red (680 ± 16 nm) and far red (λ > 710 nm) were similar except that there was elevated red fluorescence near the adaxial leaf surface relative to far red fluorescence. Fluorescence profiles were narrower in non‐infiltrated leaf samples where light scattering increased the light gradient. The fluorescence profile was broader when the leaf was irradiated on its adaxial versus abaxial surface due to the contrasting optical properties of the palisade and spongy mesophyll. Irradiation with blue, red and green monochromatic light produced profiles that peaked 50, 100 and 150 μm, respectively, beneath the irradiated surface. These results are consistent with previous measurements of the light gradient in spinach and they agree qualitatively with measurements of carbon fixation under monochromatic blue, red and green light. These results suggest that chlorophyll fluorescence profiles may be used to estimate the distribution of quanta that are absorbed within the leaf for photosynthesis.     

Phytochrome control of hypocotyl extension in light-grown Chenopodium rubrum

The regulation of hypocotyl extension in light-grown Chenopodium rubrum L. seedlings by light analogous to dense vegetation canopy shade has been monitored. Hypocotyl extension was controlled by both the quantity and quality of the actinic light. At the higher of the two background photon fluence rates which were used (10.0 μmol m⁻²s⁻¹ in the 400–700 nm waveband), increasing the proportion of phytochrome calculated to exist as Pfr resulted in greater inhibition of growth. At the lower photon fluence rate (1.0 μmol m⁻²s⁻¹ in the 400–700 nm waveband), a biphasic response was observed in which minimum inhibition was observed at intermediate photoequilibria. Although photosynthesis was not directly involved in the photomorphogenetic responses, it did play an indirect quantitative role in determining the response.     

A simple low-cost microcontroller-based photometric instrument for monitoring chloroplast movement

A new microcontroller-based photometric instrument for monitoring blue light dependent changes in leaf transmission (chloroplast movement) was developed based on a modification of the double-beam technique developed by Walzcak and Gabrys [(1980) Photosynthetica 14: 65–72]. A blue and red bicolor light emitting diode (LED) provided both a variable intensity blue actinic light and a low intensity red measuring beam. A phototransistor detected the intensity of the transmitted measuring light. An inexpensive microcontroller independently and precisely controlled the light emission of the bicolor LED. A typical measurement event involved turning off the blue actinic light for 100 μs to create a narrow temporal window for turning on and measuring the transmittance of the red light. The microcontroller was programmed using LogoChip Logo (http://www.wellesley.edu/Physics/Rberg/logochip/) to record fluence rate response curves. Laser scanning confocal microscopy was utilized to correlate the changes in leaf transmission with intercellular chloroplast position. In the dark, the chloroplasts in the spongy mesophyll exhibited no evident asymmetries in their distribution, however, in the palisade layer the cell surface in contact with the overlying epidermis was devoid of chloroplasts. The low light dependent decrease in leaf transmittance in dark acclimated leaves was correlated with the movement of chloroplasts within the palisade layer into the regions previously devoid of chloroplasts. Changes in leaf transmittance were evident within one minute following the onset of illumination. Minimal leaf transmittance was correlated with chloroplasts having retreated from cell surfaces perpendicular to the incident light (avoidance reaction) in both spongy and palisade layers.Electronic Supplementary Material Supplementary material is available for this article at     

Structural and functional properties of the coleoptile chloroplast: Photosynthesis and photosensory transduction

Recent studies have shown that guard cell and coleoptile chloroplasts appear to be involved in blue light photoreception during blue light-dependent stomatal opening and phototropic bending. The guard cell chloroplast has been studied in detail but the coleoptile chloroplast is poorly understood. The present study was aimed at the characterization of the corn coleoptile chloroplast, and its comparison with mesophyll and guard cell chloroplasts. Coleoptile chloroplasts operated the xanthophyll cycle, and their zeaxanthin content tracked incident rates of solar radiation throughout the day. Zeaxanthin formation was very sensitive to low incident fluence rates, and saturated at around 800–1000 mol m^–2 s^–1. Zeaxanthin formation in corn mesophyll chloroplasts was insensitive to low fluence rates and saturated at around 1800 mol m^–2 s^–1. Quenching rates of chlorophyll a fluorescence transients from coleoptile chloroplasts induced by saturating fluence rates of actinic red light increased as a function of zeaxanthin content. This implies that zeaxanthin plays a photoprotective role in the coleoptile chloroplast. Addition of low fluence rates of blue light to saturating red light also increased quenching rates in a zeaxanthin-dependent fashion. This blue light response of the coleoptile chloroplast is analogous to that of the guard cell chloroplast, and implicates these organelles in the sensory transduction of blue light. On a chlorophyll basis, coleoptile chloroplasts had high rates of photosynthetic oxygen evolution and low rates of photosynthetic carbon fixation, as compared with mesophyll chloroplasts. In contrast with the uniform chloroplast distribution in the leaf, coleoptile chloroplasts were predominately found in the outer cell layers of the coleoptile cortex, and had large starch grains and a moderate amount of stacked grana and stroma lamellae. Several key properties of the coleoptile chloroplast were different from those of mesophyll chloroplasts and resembled those of guard cell chloroplasts. We propose that the common properties of guard cell and coleoptile chloroplasts define a functional pattern characteristic of chloroplasts specialized in photosensory transduction.Abbreviations Ant or A antheraxanthin - dv/dt fluorescence quenching rate - Fm maximum yield of fluorescence with all PS II reaction centers closed - Fo yield of instantaneous fluorescence with all PS II reaction centers open - Vio or V violaxanthin - Zea or Z zeaxanthin     

The relationship between the redox state of Q A and photosynthesis in leaves at various carbon-dioxide,oxygen and light regimes

The response of chlorophyll fluorescence elicited by a low-fluence-rate modulated measuring beam to actinic light and to superimposed 1-s pulses from a high-fluence-rate light source was used to measure the redox state of the primary acceptor Q _A of photosystem II in leaves which were photosynthesizing under steady-state conditions. The leaves were exposed to various O₂ and CO₂ concentrations and to different energy fluence rates of actinic light to assess the relationship between rates of photosynthesis and the redox state of Q _A. Both at low and high fluence rates, the redox state of Q _A was little altered when the CO₂ concentration was reduced from saturation to about 600 l·l^-1 although photosynthesis was decreased particularly at high fluence rates. Upon further reduction in CO₂ content the amount of reduced Q _A increased appreciably even at low fluence rates where light limited CO₂ reduction. Both in the presence and in the absence of CO₂, a more reduced Q _A was observed when the O₂ concentration was below 2%. Q _A was almost fully reduced when leaves were exposed to high fluence rates under nitrogen. Even at low fluence rates, Q _A was more reduced in shade leaves of Asarum europaeum and Fagus sylvatica than in leaves of Helianthus annuus and Fagus sylvatica grown under high light. Also, in shade leaves the redox state of Q _A changed more during a transition from air containing 350 l·l^-1 CO₂ to CO₂-free air than in sun leaves. The results are discussed with respect to the energy status and the CO₂-fixation rate of the leaves.Abbreviations and symbols L 1,2 first and second actinic light beam - Q _A primary acceptor of photosystem II - q _Q Q-quenching     

CORAL PHOTOBIOLOGY STUDIED WITH A NEW IMAGING PULSE AMPLITUDE MODULATED FLUOROMETER1

A new high‐resolution imaging fluorometer (Imaging‐PAM) was used to identify heterogeneity of photosynthetic activity across the surface of corals. Three species were examined: Acropora nobilis Dana (branching), Goniastrea australiensis Edwards & Haime (massive), and Pavona decussata Dana (plate). Images of fluorescence parameters (F, F_m′, effective quantum yield, optimal quantum yield, electron transport rate, relative photosynthetic rate, and non‐photochemical quenching) allowed heterogeneity to be detected in terms of position on colony and indicated that the photosynthetic activity of polyp and coenosarc tissues responded differently to changing light for all three species. The Imaging‐PAM offers a special routine, with which images of PAR absorption (absorptivity) are obtained. In this way, for the first time it has become possible to derive images of the relative photosynthesis rate. Polyps had a lower PAR absorptivity than coenosarc tissue for A. nobilis and P. decussata, whereas G. australiensis showed the opposite pattern. Acropora nobilis showed heterogeneity along the longitudinal axis of the branch, which could be differentiated from the effect of variations in illumination across the rugose and curved surface. Diel changes were apparent and influenced the longitudinal heterogeneity along the A. nobilis branch. Images were also obtained showing the degree of photoinhibition caused by high‐light stress across a coral surface at a hitherto unobtainable level of resolution.     

Relationship between Chlorophyll a Concentration,Light Attenuation and Diving Depth of the Southern Elephant Seal Mirounga leonina

Recently, a number of Antarctic marine environmental studies have used oceanographic parameters collected from instrumented top predators for ecological and physical information. Phytoplankton concentration is generally quantified through active measurement of chlorophyll fluorescence. In this study, light absorption coefficient (K_0.75) was used as an indicator of phytoplankton concentration. This measurement, easy to obtain and requiring low electric power, allows for assessing of the fine scale horizontal structuring of phytoplankton. As part of this study, Southern elephant seals (SES) were simultaneously equipped with a fluorometer and a light logger. Along the SES tracks, variations in K_0.75 were strongly correlated with chlorophyll, a concentration measured by the fluorometer within the euphotic layer. With regards to SES foraging behaviour, bottom depth of the seal’s dive was highly dependent on light intensity at 150 m, indicating that the vertical distribution of SES’s prey such as myctophids is tightly related to light level. Therefore, change in phytoplankton concentration may not only have a direct effect on SES’s prey abundance but may also determine their vertical accessibility with likely consequences on SES foraging efficiency.     

A portable, microprocessor operated instrument for measuring chlorophyll fluorescence kinetics in stress physiology

A portable instrument for measuring chlorophyll fluorescence induction kinetics is described and examples of measurements are given. The instrument is centered around a statistically-mixed bifurcated optical fiber. One fiber branch guides the actinic light to the sample, whereas the other branch carries the emitted chlorophyll fluorescence to the photodetector. Scattered actinic light is cut out from the detector by a red interference filter. The instrument measures fast as well as slow fluorescence induction kinetics, but is particularly well designed for analyzing fast kinetics. The high time resolution and strong, variable actinic light mean that both F_o (non-variable fluorescence) and F_m (maximal fluorescence at the P-peak) are well defined. A built in microprocessor unit with attached memory stores the fluorescence induction curve and calculates key fluorescence parameters such as F_o, F_m, F_v (variable fluorescence equals F_m?F_o), F_v/F_m (the photochemical efficiency of photosystem II) and t_1/2 (half rise time from F_o, to F_m). These values are digitally displayed after each recording and they (or the whole induction curve) can be stored in a memory and later retrieved. Because of a flexible setting of the instrument it can be used with high accuracy both for optically thick leaves and for diluted suspensions of algae or chloroplasts. A simple, light weight clamp cuvette for dark adaptation of leaves has been developed. It is equipped with a gate allowing the optical fiber to be inserted without daylight reaching the dark adapted portion of the leaf. The instrument has been developed for rapid monitoring of changes in activities and organization of the photosynthetic apparatus in vivo when plants are exposed to environmental stress both in the field and in the laboratory. Examples of measurements are given for differently treated leaves of Pinus sylvestris, Salix sp., Betula verrucosa, Zea mays, Epilobium angustifo-lium and for chloroplast thylakoids isolated from Spinacia oleracea.     

Analysis of the Heat Stress Induced Quenching of Variable Chlorophyll a Fluorescence in Beet Spinach Leaves: Possible Accumulation of Oxidized Quencher P680+ under High Illumination

Effect of preheating of beet spinach leaves on chlorophyll a fluorescence yield was analyzed with the help of additional high intensity illumination pulses using a pulse modulated fluorometer. Preheating at mildly elevated temperature (35–45°C) causes a shift in the redox state of secondary donor of photosystem II, possibly due to uncoupling of phosphorylation because of thermal induced membrane disorganization and associated alkalinization of intra thylakoid space. Also, at these preheating temperatures, a rise in photosystem I catalyzed electron transfer has been shown to occur. These two effects induce rapid quenching of Chi a fluorescence, which drops even in the presence of actinic light, below the level of initial fluorescence (F_o′ monitored by the weak modulated probing light. Preheating of leaf segments induces an increase in fluorescence in the presence of dluron, which blocks electron flow between two photosystems, and thus this increases in fluorescence yield (F_o′ as monitored by weak modulated light, is not solely due to disorganization of light harvesting Chi-protein complex but also due to a shift in the redox equilibrium of the donor at the oxidizing side of photosystem II resulting in rapid reduction of Q_A the stable primary acceptor of photosystem II. In 50°C preheated DCMU treated samples, the fluorescence yield increases in weak modulated light and it approaches that of maximal steady state (F_max) level. At preheating temperature of 48°–50°C, the inactivation of enzymes in the reducing side of photosystem I, causes an impairment of the reoxidation of QA and under this condition, a strong illumination causes quenching of Chi a fluorescence. This quenching seems to arise because of accumulation of the P680⁺, the oxidized physiological donor of photosystem which is a quencher of Chi a fluorescence. This quenching depended on the pulse intensity and duration which saturates P680⁺ accumulation and is greatly manifested when water oxidation complex is damaged.     

Scanning fluorometer for the rapid assessment of pyridine nucleotide and flavoprotein fluorescence changes in tissues in vivo

This paper describes a scanning fluorometer which produces images in real time of the distribution of pyridine nucleotide or flavoprotein fluorescence at the surface of tissues in vivo. The basic difference between this device and others reported in the literature is that fluorescence changes at any selected point within the image can be quantified as they occur. We suggest that the apparatus has potential application in those areas of surgery where vascular replacement or repair is required and where it would be advantageous to have an immediate measure of the cellular response to a return of blood flow.     

The chlorophyll fluorescence quenching and changes of absorbance in pea chloroplasts

Chlorophyll (Chl) fluorescence quenching parameters were measured in dark-adapted pea leaves and chloroplasts with the purpose to find the conditions of high and low non-photochemical quenching, that would be stable during a prolonged irradiation. A PAM fluorometer was used for measuring induction curves in the range of actinic radiation of 3-35 W m-2, with an ordinary value of about 15 W m-2. The effects of various mediators, i.e., ascorbate, methyl viologen (MV), dithiothreitol (DTT) and nigericin, on the quenching process were tested. Simultaneously, the absorbance was measured during a 15-20 min period of irradiation and after the actinic radiation was turned off, i.e., in the recovery period. The pH values of chloroplast suspensions were 5.5, 6.5 and 8.0, the largest non-photochemical quenching was observed at pH of 6.5. The irradiation of chloroplasts led to an absorption decrease within the entire photosynthetically active range, attaining saturation when the fluorescence reached Fs level, and to an absorption increase during the recovery period. Absorbance changes at the maximum of red band were 10-20 %. A decrease in Chl concentration (10 %) after irradiation was found only at pH of 5.5, when the recovery time was the longest, i.e., about 60 min.     

Acclimation of Maximal Quantum Yield of Photosynthesis in the Brown Alga Alaria esculenta under High Light and UV Radiation

Abstract: Macroalgae of the upper sublittoral zone of Arctic coastal ecosystems are subjected to darkness or low light for several months during winter and have to withstand large changes in irradiance after the breakup of sea ice in the Arctic spring. Changes in photosynthetic response to high PAR (pho-tosynthetically active radiation) and UV-B radiation (UV-B) in the cold temperate brown alga A/aria esculenta were monitored with a PAM fluorometer to study photoinhibition, recovery and acclimation of maximal quantum yield of photochemistry. Plants collected in the field, as well as specimens raised in the laboratory, were exposed to various radiation conditions including different levels of PAR and UV radiation (UV-A + UV-B). Measurements of variable chlorophyll fluorescence of photosystem II revealed that the photosynthetic apparatus in A. esculenta was able to acclimate to the respective high light and UV treatments within several days. However, two different mechanisms of acclimation seem to be involved. Initially, the rate of recovery of maximal quantum yield increased after only a few exposures to high light or UV. Second, after several exposure cycles, the degree of inhibition was reduced. Data on fluorescence induction kinetics and quenching analysis showed that exposure to the respective UV radiation resulted in an increase of non-photochemical quenching, while effective quantum yield of photochemistry was hardly affected.