Transients of chlorophyll fluorescence in photosynthetic objects are often measured using short pulses of exciting radiation, which has recently been employed to capture kinetic images of fluorescence at the macroscopic level. Here we describe an instrument introducing this principle to recording of two dimensional fluorescence transients in microscopic objects. A modified fluorescence microscope is equipped with a CCD camera intensified by a micro-channel plate image amplifier. The microscopic field is irradiated simultaneously by three types of radiation: actinic radiation, saturating flashes, and pulsed measuring radiation. The measuring pulses are generated by a light-emitting diode and their duration is between 10 to 250 µs. The detection of fluorescence images (300×400 pixels, 8 bit) has a maximum time resolution of 40 ms and is gated in synchrony with the exciting pulses. This allows measuring on a background of a continuous actinic radiation up to irradiance that can elicit the maximal fluorescence yield (FM). On the other hand, the integral irradiance of the objects by the measuring radiation is very low, e.g., 0.08 µmol m-2 s-1 at 0.05 µm spatial resolution and 0.006 µmol m-2 s-1 at 4 µm spatial resolution. This allows a reliable recording of F0 even in very short time intervals (e.g., 5×80 ms). The software yields fluorescence kinetic curves for objects in user-selected areas as well as complete false-colour maps of the essential fluorescence kinetics parameters (FM, FO, FV, FV/FM, etc.) showing a two-dimensional distribution of their values. Several examples demonstrate that records of fluorescence kinetics can be obtained with a reasonable signal-to-noise ratio with all standard microscope objectives and with object sizes reaching from segments of leaf tissue to individual algal cells or chloroplasts. and H. Küpper ... [et al.].
From mature needles of white spruce, Picea glauca (Moench) Voss we isolated thylakoids capable of high rates of oxygen evolution. Oxygen-evolving activity of spruce thylakoids was labile in the absence of osmoticum and declined by 40 % during 1 h on ice, compared to a 9 % dechne observed in spinách thylakoids. We compared the relative activity in spruce and spinách of the oxygen evolving complex (OEC) and the reaction centre in Triton X-100 fractionated membranes prepared and stored for 20 or 240 h at 0 or -80 °C in media with different combinations of sucrose (0.3, 0.5 and 1.0 M) and two pH values (6.0 and 7.6). In membranes detergent- fractionated and stored at pH 7.6, photosystem 2 (PS2) activity (H2O -> DCIP) was sensitive to sucrose concentration of the medium. Spruce and spinách membranes prepared and stored in 0.3 M sucrose and pH 7.6, showed 22 and 48 % activity of their respective control membranes, freshly prepared in 1 M sucrose at pH 6.0. In contrast, in membranes prepared and stored at pH 6.0, PS2 activity was less sensitive to sucrose concentration: spruce and spinách membranes in 0.3 M sucrose showed 73 and 88 % (respectively) of the activity of membranes freshly prepared in 1 M sucrose. In both species, the degree of stimulation of DCIP photoreduction by diphenylcarbazide suggested minimal damage to the reaction centre (RC) except during preparation in 0.3 M sucrose, pH 7.6. Since the spruce RCs were not more labile than those of spinách, the extra sensitivity of spruce thylakoids in media of low sucrose concentration was likely due to extra lability of the OEC.
Winter wheat (Triticum aestivum L.) cultivars Yangmai 9 (water-logging tolerant) and Yumai 34 (water-logging sensitive) were subjected to water-logging (WL) from 7 d after anthesis to determine the responses of photosynthesis and anti-oxidative enzyme activities in flag leaf. At 15 d after treatment (DAT), net photosynthetic rate under WL was only 3.7 and 8.9 µmol(CO2) m-2 s-1 in Yumai 34 and Yangmai 9, respectively, which was much lower than in the control. Ratios of variable to maximum and variable to initial fluorescence, actual photosynthetic efficiency, and photochemical quenching were much lower, while initial fluorescence and non-photochemical quenching were much higher under WL than in control, indicating damage to photosystem 2. WL decreased activities of superoxide dismutase and catalase in both cultivars, and activity of peroxidase (POD) in Yumai 34, while POD activity in Yangmai 9 was mostly increased. The obvious decrease in the amount of post-anthesis accumulated dry matter, which was redistributed to grains, also contributed to the grain yield loss under WL. and W. Tan ... [et al.].
Nitrogen (N) starvation resulted in degreening, inhibition of photosynthetic oxygen evolution and dark respiration, reduced survival, and increased age-specific mortality in both Chlorella fusca and Chlorella vulgaris. Analysis of in vivo chlorophyll (Chl) fluorescence induction kinetics revealed the presence of N-starvation-induced changes at the level of degreened thylakoids in both species. These changes included decreased yield of the photochemistry of photosystem 2 (PS2), and a declined photosynthetic efficiency. Synthesis of secondary carotenoids represented a biochemical change in carotenogenesis that had a photoprotective effect in degreened C. fusca. This inferred photoprotection was reflected in the delayed inhibition of oxygen evolution and improved survival of C. fusca under N-starvation. The effect was further elucidated by comparison with C. vulgaris which was not able to synthesize secondary carotenoids under the same conditions.
The influence of calcium channel blockers and ionophore on Cu2+-induced changes of the photosynthetic activity of runner bean plants (Phaseolus coccineus L.) was investigated. Excess Cu2+ was applied to leaves by injection or via the roots to examine a short/local or a long time/systemic effect of this metal, respectively. The changes in fluorescence parameters indicated that the mechanism of toxic action of Cu2+ ions on the photosynthetic apparatus was only partially connected with Ca2+ or Ca2+ channels. In young plants Ca2+ diminished especially photochemical and nonphotochemical dissipative processes induced by short- and long-term influence of excess Cu2+. Blocking of Ca2+ channels did not change direct Cu2+ action on the photosynthetic activity, however, their opening distinctly intensified the inhibitory effect of the metal. After a longer accumulation peri od the effect of Cu2+ ions did not change significantly due to modified Ca2+ penetration through membranes (except that caused by La3+). Copper directly introduced into older leaves diminished only at its highest concentration the activity both of the donor and acceptor sides of photosystem 2 (PS2) connected with Rfd decrease and increase of LNU. A similar effect was observed also after a long-term Cu2+ action, but disturbances on the acceptor side of PS2 were observed only at a higher Ca2+ content in the nutrient solution. Ca2+ ions, particularly after openning of channels, intensified direct inhibitory Cu2+ action on the photosynthetic activity expressed by decreased values of Fv/F0 and Rfd. Lanthanum and verapamil, at a lower Ca2+ content in the medium, decreased the photosynthetic activity of Cu2+-treated plants. This effect was also seen after additional Ca2+ supply to the leaves. and W. Maksymiec, T. Baszyński.
Cuttings of five Populus alba clones (S18 F1-26, Al29 F8-35, J3 F1-4, GU1 F16-36, PO9 F21-88), Populus euphratica, and Populus×euramericana (I-214) were submitted during 45 d to regular watering with NaCl solutions of electrical conductivity of 7 and 14 dS m-1. Chlorophyll a fluorescence in response to the salinity stress was assessed, using F0 and Fv/Fm. Differences in reaction to the salt were found in P. alba clones, F0 and Fv/Fm being the fluorescence parameters used to check out this stress. Minimal constant fluorescence of dark-adapted plants (F0) showed a better correlation with the disease index exhibited by plants and also with salinity dose than the parameter Fv/Fm. Some of the P. alba clones showed the same behaviour, assessed through fluorescence parameters, as P. euphratica, which was previously defined as salt tolerant, while the rest exhibited the same characteristics as I-214, which was very sensitive. and H. Sixto, I. Aranda, J. M. Grau.
The photosynthetic pigments and photochemical efficiency of photosystem 2 (PS2) were studied in four constitutive species (Achillea millefolium L., Festuca pseudovina Hack. ex Wiesb., Potentilla arenaria Borkh., and Thymus degenianus Lyka) of a semiarid grassland in South-eastern Hungary. Every species displayed typical sun-adapted traits and substantial plasticity in the composition and functioning of the photosynthetic apparatus. The contents of chlorophylls (Chls) and carotenoids (Cars) on a dry matter basis declined from May to July, however, the amount of total Cars on a Chl basis increased. This increase was the largest in Potentilla (48 %) and the smallest in Achillea (14 %). The pool of xanthophylls (VAZ) was between 25 % and 45 % of the total Car content and was larger in July than in May. The content of β-carotene increased by July, but lutein content did not change significantly. The Chl fluorescence ratio Fv/Fm was reduced by 3-10 % at noon, reflecting the down-regulation of PS2 in the period of high irradiance and high temperature. The occurrence of minimal values of ΔF/Fm' showed close correlation to the de-epoxidation rate of violaxanthin. Hence in natural habitats these species developed a considerable capacity to dissipate excess excitation energy in the summer period in their photosynthetic apparatus through the xanthophyll cycle pool and a related photoprotective mechanism, when the photochemical utilization of photon energy was down-regulated. and S. Veres ... [et al.]
Thylakoid membranes (TM) of the cyanobacterium Synechococcus elongatus were exposed for 30 min to the influence of 0, 10, 100, and 1 000 mM CdCl2 (= Cd0, Cd10, Cd100, and Cd1000). Cd10 and Cd100 caused some increase in activity of photosystem 2, PS2 (H2O → DCPIP), while distinct inhibition was observed with Cd1000. We also observed a similar effect when measuring oxygen evolution (H2O → PBQ + FeCy). Chloroplasts of spinach (Spinacia oleracea L.) were incubated for 30 min with 0, 15, 30, and 60 mM CdCl2 (= Cd0, Cd15, Cd30, and Cd60). All concentrations studied inhibited the PS2 activity, the effect being stronger with increasing concentration of Cd2+. The photosynthetic oxygen evolution activity was also influenced most distinctly by the highest concentration employed, i.e. Cd60. Electrophoretic analysis of the protein composition of cyanobacterium TM showed chief changes in the molecular mass regions of Mr 29 000 and 116 000, while with spinach chloroplasts the most distinct differences were observed in the regions of Mr 15 000 and 50 000. Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) activity in cyanobacterial spheroplasts still remained on the 40 % level in the case of Cd1000, but it decreased down to approx. 2.5 % in the Cd60 sample of spinach chloroplasts. and M. Nováková, E. Matějová, D. Sofrová.
The effects of drought and the diurnal changes in photosynthetic electron transport were studied in non-nodulated plants of Casuarina equisetifolia. The induction of fluorescence showed a slightly higher I step in water-stressed than control plants, and the time from the start of irradiation to the P step of induction was significantly shortened by drought. The quantum efficiency of photosystem 2 (PS2) in the dark-adapted state (Fv/Fm) was generally not affected by drought, whereas it decreased during the central hours of the day. The decrease in quantum yield of PS2 electron transport (Φ2) in water-stressed plants was associated with decreases in the photochemical efficiency of open (oxidised) PS2 centres (Fv'/Fm') and increases in non-photochemical quenching (qN) rather than with increased closure of PS2 centres (lowered photochemical quenching, qP). In contrast, the changes in quantum yield of electron transport during the day were related to changes in qP rather than in Fv'/Fm'. When chlorophyll fluorescence was measured at the same irradiance during the day, a greater qN was observed at the end of the drying cycle than after watering, and early and late in the photoperiod than in the central hours of the day. The greater qN at the beginning and end of the day did not prevent an increase in energy not used photochemically nor dissipated non-photochemically. Drought did not affect this excess of photon energy. and R. Martínez-Carrasco, J. Sánchez-Rodriguez, P. Pérez.
Changes in the content of pigments and rate of photosynthesis in Azolla microphylla Kaulf. fronds were measured during growth under solar and ultraviolet-C (UV-C) supplemented solar radiation. Maximum content of total chlorophyll (Chl) was observed on the 13th day (termination of the experiment) of treatment in both control and treated plants. The treated plants had significantly lower total Chl and carotenoid contents than the control plants during the 1st day of growth. After the 4th day of exposure to UV-C supplemented solar radiation, the Chl and carotenoids accumulation increased in treated plants, so that the pigment concentration in the treated fronds was nearer to the control values after the 13th day of treatment. Significant increase in UV absorbing pigments, anthocyanins, and flavonoids was observed at the 13th day of treatment. In spite of the roughly similar photosynthetic pigment concentration, the photosynthetic activity measured as the rate of electron transport at photosystem 2 was only 65 % of the control values after 13 d of UV-C exposure. and M. Jayakumar ... [et al.].