Chlorophyll Fluorescence Induction Kinetics Research Articles

The effect of ozone fumigation over one season on photosynthetic processes of Quercus robur seedlings

summary Quercus robur saplings were exposed to elevated O3 (80 nmol mol‐1) in hemispherical glasshouses for one growing season in order to assess the effects of this pollutant on photosynthesis. Measurements of chlorophyll fluorescence induction kinetics in mid‐summer showed that plants exposed to both control O3 (20 nmol mol−1) and elevated O3 treatments experienced significant photoinhibition of photosynthesis. Elevated O3 increased photoinhibition. At the end of the summer, photosynthetic CO2 uptake, measured at either light saturation or under light‐limiting conditions, was not significantly different between plants from the two O3 treatments. Although the maximum rate of carboxylation velocity, as estimated from the response of CO2 uptake to intercellular CO2 concentration, the maximum quantum efficiency and the maximum rate of electron transport measured in vivo were decreased by O3 exposure, the responses differed between plants and, overall, the decreases were not statistically significant. The results suggest that O3 levels that are at present reached for a few days during photochemical episodes in the summer are not sufficient to cause measurable lasting damage to photosynthetic CO2 uptake in oak saplings assessed over one growing season.

Effect of PS II-herbicides on algae grown in ponds and measured by the 10 µs resolved chlorophyll fluorescence induction kinetics

Algae grown under natural conditions in ponds have been investigated by measuring the chlorophyll fluorescence induction kinetics with a maximum time resolution of 10 μs. The initial phase of the intensity F(t) can be described by a fit-function including two exponential functions with the time constants T a1 = 0.8-1.7ms and T a2 = 10-60 ms. From the corresponding amplitudes F a1 and F a2 of these exponential functions, the parameter B = F a1 /(F a1 + F a2 ) is derived. Comparative measurements on original pond water and on samples after centrifugation and filtration prove a significant increase in B after an application of PS II-herbicides. Further measurements on centrifuged samples with terbuthylazine (1-200 μg/l) and atrazine (10-200 μg/l) show first effects with concentrations of 1-10 μg/l and a continuous increase in B with the PS II-herbicide concentration. As copper ions do not yield significant effects on the induction kinetics in the range 0-50ms, B indicates preferably the herbicide action on QB. The reversibility of the B-increase induced by terbuthylazine demonstrates that no accumulation of this herbicide occurs in the algae.

Application of the Karlsruhe CCD-OMA LIDAR-Fluorosensor in Stress Detection of Plants

Summary The Karlsruhe CCD-OMA LIDAR-Fluorosensor (excitation: cw HeNe laser, 632.8 nm; detection:complete chlorophyll fluorescence spectra between 650-800 nm within 10 ms) was developed in a joint project by the Technical University of Budapest and the University of Karlsruhe for a non-destructive stress detection of plants. The computer-aided fluorosensor permits to measure the full fluorescence spectra at 8 different time intervals during the chlorophyll fluorescence induction kinetics, from which the the variable chlorophyll fluorescence ratio Rfd as plant vitality index can be calculated. The position of the chlorophyll fluorescence emission maxima of green leaves are found in the red region near 690 nm and the far-red region near 735 nm. The wavelength position of the two maxima remained unchanged during the light-induced fluorescence induction kinetics (Kautsky effect). The absolute fluorescence intensity and the ratio of the two chlorophyll fluorescence bands (F690/F735) depended on the chlorophyll content and the photosynthetic activity of a leaf as has been demonstrated here with the CCD-OMA LIDAR fluorosensor. With increasing chlorophyll content of the leaf the fluorescence intensity of the red band near 690 nmdecreased, whereas that of the far-red band near 735 nm remained constant or slightly increased. Consequendy, the fluorescence ratio F690/F735 increased with decreasing chlorophyll concentration of leaves (curvi-linear relationship: y = a·x-b). Changes in the fluorescence ratio F690/F735 can be taken as an indicator of long-term stress affecting the chlorophyll content of leaves. The inverse relationship between the intensity of the chlorophyll fluorescence and the rate of photosynthesis (Kautsky effect) can be used for detecting, via Rfd-values, a short-term damage to plants, which affects photosynthetic activity, but does not yet decrease the chlorophyll content of the leaf. Damage or stress is indicated by low Rfd-values (ratio fluorescence decrease) or low Ap-values (stress adaptation index). Examples are shown for different types of damage and/or stresses (water stress, forest decline phenomena, application of a herbicide inhibiting photosynthesis, and biological stress, e.g. damage by mites). The Karlsruhe CCD-OMA LIDAR-Fluorosensor proved to be a valuable tool for fast detection of stress to plants in the laboratory, but can also be applied for ground-truth control measurements during remote sensing of the state of health of terrestrial vegetation.

Direct observation of herbicide action in algae using 10 µs resolved chlorophyll fluorescence induction kinetics

Direct observation of herbicide action in algae using 10 µs resolved chlorophyll fluorescence induction kinetics

FREEZING STRESS AND OSMOTIC DEHYDRATION IN FUCUS DISTICHUS (PHAEOPHYTA): EVIDENCE FOR PHYSIOLOGICAL SIMILARITY1

ABSTRACTThe effects of osmotic dehydration and freezing on photosynthesis were studied in the brown alga Fucus distichus L. The data indicated that F. distichus exhibits similar physiological responses to both osmotic dehydration and freezing stress and that these responses resemble those in the literature for the effect of desiccation in air. Both stresses inhibited light‐limited (Psubsat) and light‐saturated (Pmax) photosynthesis measured immediately after plants were reimmersed in seawater. The degree of initial inhibition and subsequent recovery of photosynthesis were proportional to the severity of the dehydration or freezing treatment. Psubsat and Pmax recovered completely from osmotic dehydration for 3 h in 200% and 3 hr at – 10°C, but recovery was only partial following 3 h in 300%o or 3 h at – 15°C. In most cases, recovery was complete within 2 h following dehydration, with little further recovery occurring between 2 and 24 h posttreatment. No time‐dependent recovery occurred following severe freezing. Observations using the vital stain fluorescein diacetate suggested that the lack of complete recovery might be due to severe damage or death of a proportion of cells in the thallus. There were no clear effects of either osmotic dehydration or freezing on dark respiration (Rd), although Rd was stimulated in all emersed treatments (frozen plants and 5° C controls) immediately following reimmersion. Measurement of chlorophyll fluorescence induction kinetics indicated that both osmotic dehydration and freezing reduced the ratio of variable to maximum florescence (Fv/Fm), indicating a decrease in the quantum efficiency of photosystem I. Based on these data, we suggest that there are common cellular and physiological components involved in the response of fucoid algae to a range of water stresses. This hypothesis was supported by experiments that showed that osmoacclimation in hyperosmotic seawater (51%o)for 2 weeks increased the ability of F. distichus to recover from freezing at – 15° C. During acclimation, mannitol content increased under hyperosmotic conditions and decreased under hypoosmotic conditions. Changes in plasma membrane integrity, determined by fresh weight: dry weight ratio, and amino acid release following freezing indicated an increasing gradient of freezing tolerance from low to high salinity. However, none of these physiological changes fully explained the marked increase in the freezing tolerance of photosynthesis observed in plants acclimated under hyperosmotic conditions.

Chlorophyll fluorescence and delayed fluorescence as potential tools in remote sensing: A reflection of some aspects of problems in comparative analysis

Induction kinetics of chlorophyll fluorescence (the Kautsky effect) and delayed fluorescence (DF) of some cold climate species were studied in both laboratory and in situ experiments to assess their vlaue as indicators of plant health. Experiments included studies of natural variability of fluorescence in connection with the seasonal leaf maturation process, as well as the influence of environmental stress factors, such as ozone. It was found that parameters of induction curves (normalized variable fluorescence, time of its half decrease) could be used as indicators of plant health. No significant differences have been elicited between the information content of the Kautsky effect and induction curves of DF as tools for remote sensing. It is suggested that measurements of the single decay curves of DF are preferable for remote estimation of photosynthetic capacity.

Studies on the constancy of the blue and green fluorescence yield during the chlorophyll fluorescence induction kinetics (Kautsky effect).

Blue (F450) and green (F530) leaf fluorescence were studied together with the red chlorophyll fluorescence (emission maxima F690 and F735) during light-induced chlorophyll fluorescence induction kinetics (Kautsky effect) in predarkened leaves of wheat (Triticum aestivum L.) and soybean (Glycine max L.). The intensity of the red chlorophyll fluorescence decreased from maximum fluorescence Fm to steady-state fluorescence Fs, and the fluorescence ratio F690/F735 decreased by about 10% from Fm to Fs. However, blue and green fluorescence intensities remained constant throughout the measuring time. Consequently, the ratio of blue to red fluorescence (F450/F690) increased during chlorophyll fluorescence induction kinetics, whereas the ratio of blue to green fluorescence (F450/F530) remained unchanged within the same period. The knowledge of these ratios will be a prerequisite for the interpretation of remote sensing data from terrestrial vegetation.

Investigations on the photosynthetic system of spruce affected by forest decline and ozone fumigation in closed chambers

Investigations using chlorophyll fluorescence induction kinetics provide the parameters R fd , L and A p to characterize different specifications of the photosynthetic system (PS). The application of ozone, with concentrations between 100 μg m −3 and 2000 μg m −3 to spruce in closed chambers yields a reduction of R fd between 6% and 23% for the current year's needles, which indicates a reduced potential photosynthetic capacity. Further measurements on the current year's needles of spruce of the damage classes SO/S1, S2 and S3 show also a reduction of R fd of between 7% and 14% in 2 successive years. In addition, the parameter L increases for damaged trees by between 11% and 49%, indicating a change of the chlorophyll content and of the internal energy distribution between PSI and PSII. As no effect of L can be observed with ozone fumigation, it is concluded that the change of some specifications of the PS can be simulated well by ozone fumigation (e.g. R fd ) while other specifications cannot (e.g. L).

Photoinhibition and Light Acclimation in Seedlings of Bischofia javanica, a Tropical Forest Tree from Asia

The aim of this study was to examine the potential for light acclimation in shade grown seedlings of Bischofia javanica Blume. The seedlings were grown under simulated forest shade light (40 μmol m -2 s -1 ), and after transfer to a higher light level (1200 μmol m -2 s -1 ). chlorophyll fluorescence induction kinetics, net photosynthesis, and changes in leaf chlorophylls and leaf anatomy were examined in leaves that were fully developed prior to the transfer. The low-light (LL) leaf displayed photoinhibition immediately after transfer to high-light (HL) (...)

Measurement of the chlorophyll fluorescence induction kinetics with a 10 �s time resolution and its application in the forest decline research

Measurement methods are described which determine the initial phase of the fluorescence induction kinetics with a maximum time resolution of 10 microseconds simultaneously for the two fluorescence components F685(t) and F730(t) selected by filters at the wavelengths 685 nm and 730 nm, respectively. The excitation light provided by a He-Ne laser (632.8 nm) is switched on within 0.3 microseconds (maximum intensity Ie = 12 mW/cm2). Fo, Fp, and Fs, the initial-, peak-, and steady-state intensity and the initial value Ro of the ratio R(t) = F730(t)/F685(t) can accurately be determined as well as the initial time derivative Fo* of the fluorescence intensity. Fo and Fo* are related to the quantum yield phi a of the antenna and to the photochemical quantum yield phi pc, respectively. Spruce, oak, birch, poplar, and soy bean show a decline of R(t) from Ro to a first minimum Rb at some 10 ms which has a similar value as the second minimum Rp in the time range of seconds. Furthermore, the initial value Ro and the steady-state value Rs of R(t) are also very similar. Measurements on spruce with water deficiency and with varying excitation light intensity Ie show effects on the initial phase of the fluorescence induction kinetics. Further measurements on spruce of different damage classes indicate that for the current year's needles the ratio Fp/Fo is the most sensitive parameter to differentiate between the damage classes and that Fo/Fs and Ro/Rb are also affected. As demonstrated by measurements on leaves of soy beans, the initial decrease of R(t) from Ro to Rb originates from a change of the fluorescence spectrum because no change of the leaf transmission can be observed in the time range between 10 microseconds and 1 ms.

Effect of UV-B Radiation on Leaf Optical Properties Measured with Fibre Optics

Changes in the internal light microenvironment in leaves of plants of Brassica campestris L. cv. Emma, B. carinata L., and Medicago saliva L. cv. Armour in response to exposure to UV-B (UV-B, 280–320 nm) radiation were measured using a fibreoptic microprobe. Plants were exposed for 2 weeks either to high visible light or to supplemental ultraviolet-B radiation. The spectral regime (400–700 nm; PAR) was measured either midway through the leaf palisade or the spongy mesophyll. After exposure to UV-B radiation leaves of Brassica campesiris attenuated transmitted light more than the controls. At the same time both forward and back scattered light increased in the palisade and spongy mesophylls. In contrast, UV-treatment of Medicago saliva leaves increased light transmission into the palisade, while the back scattered component showed little change. Leaves of cariiwla showed little change in response to UV. Other responses to UV-B radiation included increases in leaf thickness, decreased total chlorophyll content, and changes in UV-B screening pigments and chlorophyll fluorescence induction kinetics. Brassica campestris was most sensitive to exposure to enhanced levels of UV-B radiation, whereas leaves of B. carinata were the least sensitive. Our data indicate that exposure to UV-B radiation altered the light microenvironment within leaves of the species different ways. These changes appeared to be caused by alterations in pigment content and leaf anatomy. In turn, the altered distribution of PAR within the leaf could influence photosynthesis.

A device for the determination of the microsecond component of the in vivo chlorophyll fluorescence induction kinetics

A device for the measurement of the in vivo chlorophyll fluorescence induction kinetics at the wavelengths 685 nm and 730 nm is described. The excitation light provided by a He-Ne laser is switched by an acousto-optic modulator (Bragg cell) which achieves a rise time of the excitation light intensity Ie of 0.3 mu s. A combination with an additional mechanical shutter ensures that Ie is constant within 11% during the total measuring time of 5 min. Data acquisition is accomplished by a transient recorder with a time resolution of 10 mu s and a computer with a sampling rate adjusted between 5000 s-1 and 5 s-1. The initial fluorescence intensity F0, the inflection point Ip (during the rise of the fluorescence at the beginning), the maximum intensity Fp and the final steady-state value Fs can be evaluated with high accuracy, so additional information is provided to estimate the efficiency of the photosynthetic system.

Protein phosphorylation and control of excitation energy transfer in photosynthetic purple bacteria and cyanobacteria

The function of phosphorylation of light-harvesting polypeptides is well characterised in chloroplasts of green plants, but the prokaryotic cyanobacteria and purple photosynthetic bacteria have quite different light-harvesting polypeptides whose structure and function cannot be controlled in precisely the same way. Nevertheless, cyanobacteria show light-dependent phosphorylation of membrane polypeptides associated with photosystem II and with the light-harvesting phycobilisome, and purple bacteria show light-dependent phosphorylation of low molecular-weight chromatophore membrane polypeptides. In both cases membrane protein phosphorylation is associated with functional changes observed by chlorophyll fluorescence spectroscopy or chlorophyll fluorescence induction kinetics. Here we report on our recent protein sequence and other data concerning the identities of these phosphoproteins. We also discuss the significance of these findings for regulation by protein phosphorylation of photosynthesis in prokaryotes.

Positive selection for sensitivity of Chlamydomonas reinhardtii to Photosystem II-inhibiting herbicides

We have developed a method for the strong positive selection of 3-(3,4-dichlorophenyl)-l,l-dimethylurea (DCMU or diuron) -sensitive mutants of the unicellular green alga Chlamydomonas reinhardtii . The method involves plating cells in the light on medium containing acetate with carefully balanced concentrations of diuron and metronidazole. We have used this method for the selection of sensitive revertants of diuron-resistant strain Dr2. Among the strains that survive the selection, the spectrum of mutant phenotypes is diverse. These include reduced or absent photosynthesis, altered pigment content or chlorophyll fluorescence induction kinetics, as well as increased herbicide sensitivity. Among the more sensitive strains characterized in detail were five with increased affinity of diuron binding to the thylakoid membranes. None of thesemutants is as sensitive as wild type, and so none is an exact revertant of mutant Dr2. AH of these mutants showed the non-mendelian inheritance pattern characteristic of chloroplast DNA. Thus, the results extend the previous conclusion that the Photosystem II herbicide binding site is under the exclusive control of the chloroplast genome.

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 Fo (non‐variable fluorescence) and Fm (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 Fo, Fm, Fv (variable fluorescence equals Fm−Fo), Fv/Fm (the photochemical efficiency of photosystem II) and t1/2 (half rise time from Fo, to Fm). 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.

Gabaculine Inhibition of Chlorophyll Biosynthesis and Nodulation in Phaseolus lunatus L.

Gabaculine (3-amino-2,3-dihydrobenzoic acid) was an inhibitor of in vivo chlorophyll biosynthesis in lima bean (Phaseolus lunatus L. cv Henderson). When applied to roots of 9-day-old plants, 10 micromolar gabaculine was sufficient to terminate biosynthesis of new chlorophyll. The trifoliolate leaves which emerged after gabaculine treatment were yellow. Gabaculine-treated plants had slightly lower dry weights; yet, overall plant size showed very little change. Chlorophyll fluorescence induction kinetics and CO(2) exchange measurements were used to monitor both immediate and long-term effects of gabaculine on photosynthesis. A lowered rate of the decline from the maximum level of fluorescence was observed after 10 hours for nitrate-supplemented plants, and all treated plants showed a slightly increased level of original fluorescence after 6 days. No change was observed in the rate of photosynthesis by unifoliolate leaves. The trifoliolate leaves, though not able to photosynthesize, were able to continue respiration. This suggested that heme biosynthesis for mitochondrial cytochromes was not abolished. In untreated lima bean, root nodules were induced by Rhizobium sp. 127E15. Following gabaculine treatment, root nodules formed, but were largely ineffective in nitrogen fixation. Nodule dry weight, nitrogen fixation activity, and leghemoglobin content were decreased by gabaculine.

Analysis of chlorophyll fluorescence induction kinetics exhibited by DCMU-inhibited thylakoids and the origin of α and β centres

Analyses of room-temperature chlorophyll fluorescence curves from DCMU-inhibited thylakoids were used to investigate the proposed PS II structural heterogeneity of α and β centres. The kinetics of the area growth curves, representative of Q A photoreduction, could be modified in the presence of DCMU by exogenous electron acceptors and by added reductants of the PQ pool. The effect of altered DCMU levels (range 0.2–100 μM) on the induction curve kinetics was to modify preferentially the slow-β component, while having only a very small effect on the total variable fluorescence yield. Over the DCMU concentration range used, the unnormalized area of the induction curve ( A max) decreased with increasing herbicide concentration by approx. 45%, indicating that less quanta were required to reduce Q A. It was found that the dark reoxidation of Q A in the presence of DCMU and Ant 2p after a light pretreatment regenerated the slow kinetic component. When chlorophyll fluorescence emission at 685 and 731 nm was measured, no difference was observed in the kinetics of the induction curve. The analysis of PS II-enriched, oxygen-evolving membranes indicated the presence of both the fast and slow kinetic components, although this type of preparation showed a modified fast phase. The above observations led to the conclusion that several of the previously proposed characteristics of PS II α and PS II β centres do not hold and that a type of PS II heterogeneity involving different degrees of DCMU inhibition is sufficient to explain many of the observations made.

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.

Evidence for the role of the light-harvesting chlorophyll [formula omitted] protein complex in photosystem II heterogeneity

Analyses of chlorophyll fluorescence induction kinetics from DCMU-poisoned thylakoids were used to examine the contribution of the light-harvesting chlorophyll a b protein complex (LHCP) to Photosystem II (PS II) heterogeneity. Thylakoids excited with 450 nm radiation exhibited fluorescence induction kinetics characteristic of major contributions from both PS II α and PS II β centres. On excitation at 550 nm the major contribution was from PS II β centres, that from PS II α centres was only minimal. Mg 2+ depletion had negligible effect on the induction kinetics of thylakoids excited with 550 nm radiation, however, as expected, with 450 nm excitation a loss of the PS II α component was observed. Thylakoids from a chlorophyll- b-less barley mutant exhibited similar induction kinetics with 450 and 550 nm excitation, which were characteristic of PS II β centres being the major contributors; the PS II α contribution was minimal. The fluorescence induction kinetics of wheat thylakoids at two different developmental stages, which exhibited different amounts of thylakoid appression but similar chlorophyll a b ratios and thus similar PS II:LHCP ratios, showed no appreciable differences in the relative contributions of PS II α and PS II β centres. Mg 2+ depletion had similar effects on the two thylakoid preparations. These data lead to the conclusion that it is the PS II:LHCP ratio, and probably not thylakoid appression, that is the major determinant of the relative contributions of PS II α and PS II β to the fluorescence induction kinetics. PS II α characteristics are produced by LHCP association with PS II, whereas PS II β characteristic can be generated by either disconnecting LHCP from PS II or by preferentially exciting PS II relative to LHCP.

Chloroplast Biogenesis, Its Inhibition and Modification by New Herbicide Compounds

Biogenesis and development of chloroplasts are summarized in the light of new findings; the interactions of different groups of photosynthetic herbicides with chloroplast development are indicated. The effect of two new active ingredients, mevinolin* and sethoxydim **, on chloroplast modification and biogenesis are described. Mevinolin blocks the cytoplasmic HMG-CoA reductase and sterol accumulation and modifies chloroplast differentiation via the formation of a sun-type chloroplast. Sethoxydim inhibits leaf growth in grasses and gramineous crop plants and strongly affects chloroplast biogenesis and differentiation at all stages of development from proplastids via prochloroplasts to young and mature chloroplasts. This applies to thylakoid formation and multiplication, grana formation as well as chloroplast division and chlorophyll and carotenoid accumulation. The chlorophyll fluorescence induction kinetics (Kautsky effect) indicate that photosynthesis is not affected. At higher doses sethoxydim stops all growth responses of maize plants and induces the accumulation of anthocyanins.