Fluorescence induction measurement

Chemiluminescence of oxidized luminol has been the basis of several lumino-metric methods of estimation of TAC (Table 1). The mostcommon is to measure the induction time of the reaction. Often the chemiluminescence is first induced by an oxidant and then attenuated by addition of a sample, and the time to recover the initial fluorescence is measured. The enhanced chemiluminescent assay introduced a decade ago is based on the oxidation of luminol by perborate or by hydrogen peroxide in a reaction catalyzed by horseradish peroxidase. Enhancement (and stabilization) of luminescence is achieved by addition of p-iodophenol. The original procedure used a commercial reagent kit (ECL Anti-oxidant Detection Pack... 

From the measured kinetics of P700 photooxidation and area over fluorescence induction we determined the functional chlorophyll antenna size of PSI and PSII, respectively [Melis 1989], for the control and Chi Mess mutant (Table 3). 

D.-P. Hader, H. Herrmann, J. Schafer, R. Santas (1997). Photosynthetic fluorescence induction and oxygen production in two Mediterranean Cladophora species measured on site. Aquat. Bot., 56, 253-264. 

During fluorescence induction, a first transient, i, is observed that corresponds to the oxidation of an intermediate pool and Q by PS I (11). This fluorescence transient can be measured easily for chloroplast suspensions in the presence of inhibitors (Figure 1). From the values observed on the induction curves, the corresponding A (J) - A cj) / A jjj x were cal-... 

Fluorescence induction experiments measure changes in fluorescence yield arising from functional PS II. Fluorescence was measured at 690 nm with excitation at either 440 or 600 nm in dark-adapted cells treated with either dichlorophenyl dimethylurea... 

Very pure preparations of heterocysts of Nostoc ATCC 29150 lacked PSn activity as measured by oxygen evolution and by fluorescence induction. The absence, in this strain, of phycobiliproteins is consistent with inactive PSII. Others have reported phycobiliproteins in some strains of cyanobacteria (7,8) thus, their presence may depend on the strain or on the conditions of growth. Although there is no functional PSn, the proteins of the complex are present in heterocysts, and fluorescence spectra indicate that chlorophyll may be bound to CP47. 

We have previously pointed out that, under the appropriate conditions, the sigmoidally shaped fluorescence induction curves should also be observed when the PS II reaction centers are partially closed by short, pulsed light flashes and when the fluorescence yield is measured with a weak probe light flash delivered at some time 6t (30 - 100 ps) after the variable - intensity pump flash (3). This follows from the assumption that under either steady-state or flash-excitation conditions, the fraction of closed reaction centers q should depend simply on the number of photons absorbed by PS II In both cases. However, using pump flashes of less than 1 /is in duration, the fluorescence induction curves measured by the pump-probe technique have been shown to be exponential in shape [3.4]. Similar obsenrations have been made by Mauzerall and his co-workers [5.6] who concluded that the probability of escape of an exciton from a PS II unit with a closed reaction center to a unit with an open one. is less than 0.25 and that the apparent optical cross-section of PS II with open and closed traps is constant to within + 10 % [7]. The exponentiaiity of the pump-probe fluorescence Induction curves implies that the variable fluorescence Fy = (F[l ] - Fo)/(Fmax " Fq) is proportional to q under these conditions, where 1 represents the fiuence of the pump flash expressed in units of incident photons/cm. ... 

Fig. 1 (left) Top Fluorescence induction cunre measured with the laser pump flash Pi, and 2 is xenon probe flash arriving 100 /js after the pump flash (no DCMU). F2 is the probe flash fluorescence yield reiative to the yield in the absence of Pi. Bottom conventional fluorescence induction curve measured under steady-state conditions using a He-Ne iaser. 

The inhibition of the electron flow at site 2 is also shown by analyzing the data of Chi a fluorescence induction curves. The initial slope of Ghl a fluorescence (upto 1 s of illumination) that reflects the reduction of Q, in spinach leaf discs as a function of [H002 ] concentration was accelerated (data not shown). These measurements indicate the inhibitory effect of HQ02 on the electron flow of PS II. Addition of bicarbonate reversed the H002 effect it slowed down the rise of the fluorescence (up to one second of illumination) and decreased the ratio of variable to constant fluorescence as shown in Fig. 4. This further confirms the role of HCD3 in reversing the... 

At the measurements of fluorescence induction using double beam fluorimeter, switching off the actinic light causes the decay of fluorescence intensity to the level excited by low intense measuring beam. Earlier the study of dark relaxation kinetics of variable fluorescence was done on isolated chloroplasts and their fragments (4-7). 

Switching off the actinic light and immediate illumination of the leaf by light 1 (A>710 nm) induces fast fluorescence dec to initial level. Light 1 accelerates the dark relaxation at any phase of induction (Fig, la). Thus, dark relaxation of variable fluorescence at measured conditions reflects mainly Q reoxidation, and the fast component of relaxation is associated with the electron... 

The barley chloroplasts were isolated as reported by Beauregard al (1987) (4). The alga Dunaliella tertiolecta was grown in the culture medium described by Harrison et al (1980) (5). Fluorescence induction curves were obtained by using an integrating sphere, as reported earlier (6). The electron acceptors were added 1 min. prior to the fluorescence measurements. The Fq fluorescence yields were evaluated by using our least square regression method (3). 

We simultaneously measured the oxygen evolution and fluorescence induction kinetics of green algae. The experimental results are compared with computer simulations. 

Energy transfer between PSII units was determined by analysing the normalised variable fluorescence as a function of the area growth above the fluorescence induction curve (12), measured in the presence of DCMU and hydroxyl amine. 

In the PPFD range of 0-500 pmol m" s the R2K1 mutant appears to be somewhat less sensitive to photoinhibition than do the other mutant R2S2C3 and the wild type R2 (Table 1). This is shown both by the 77K fluorescence induction kinetics data O2 evolution measured as pmol Or... 

Attached leaves of pumpkin (Cucurbita pepo L.) were photoinhibited at 750, 1500 or 2500 umol PAR m 2s either at room temperature (RT) or at 1 0, in saturated humidity. After the treatment, different photosynthetic parameters were measured either from leaf discs (fluorescence induction at 77 K, apparent quantum yield of oxygen evolution), or from thylakoids isolated from treated leaves (electron transfer activities, fluorescence induction in the presence of DCMU or FeCN, atrazine binding). 

The mutant, B6, was obtained following FdUrd treatment of wild type cells (6). Genetic analysis indicated that B6 is a chloroplast mutant (Roitgrund and Mets, in preparation). Measurements of fluorescence induction showed typical fluorescence rise kinetics as expected for cells possesing an active photosystem II, but impaired in the oxidation of reduced plastoquinol (Fig. i). To test the possibility that the cytochrome subunits are present but inactive, cyt. bjf components were assayed by TMBZ heme staining and by immunoblotting with specific antibodies. 

Fluorescence induction was measured at room temperature with a modified Aminco spectrophotometer. Actinic light of uniform field was provided in the blue-green region of the spectrum. 

Fluorescence emission at 685 nm was detected at right angles to the direction of the actinic beam. Integration of the area over the fluorescence induction curve and semilogarithmic plots were analysed according to Melis and Homann (4,5). The reaction mixture contained 50 mM Tricine, pH 7.8, 400 mM sucrose, 10 mM NaCl, 5 mM MgCl and membrane vesicles (5/iM Chl/ml). Fluorescence was measured in the presence of 20 /zM DCKU and after 5 min for dark adaption. 

The kinetics of the variable fluorescence of the different fractions show that the BS fraction needs the shortest time to reach half maximal rise in variable fluorescence, and also have the largest kinetic constant for the increase of the area over the fluorescence induction curve (Table 1). From the data of Table 1 one can conclude that the functional antenna size of all the three fractions originating from B3 differ in the same order as was found by measuring the light saturation curves (2). 

Fluorescence induction kinetics was measured at room temperature with a modified Aminco spectrophotometer in the presence of 20 mM DCMU after 5 min of dark adaptation. Relative rate of pheophytin anion photoaccumulation in PSII was estimated from the analysis of the fluorescence quenching curve in the presence of 20 mM dithionite (in 50mM Tricine, pH 7.8). 

TABLE 1. Comparison of the fluorescence induction curve parameters measured from thylakoids isolated from spinach grown at... 

TABLE 2. Fluorescence induction curve parameters measured on different thylakoid membrane fragments isolated from spinach grown under different light conditions. 

When dealing with leaves such a condition is not usually met as the pigment concentrations encoutered in vivo are usually very high. The kinetic analysis is further complicated because the photosynthetic apparatus will adapt to the light conditions prevailing at different depths in the leave. However the fluorescence from the uppermost cell layers can be measured selectively using an appropriate choice of the excitation and emission wavelengths (3). Fig. 1 shows a typical fluorescence induction curve measured from a DCMU inhibited leave. The fluorescence reached its maximal value in essentially 100 msec. Contrary to what has been observed with thylakoids the fast rise was not followed by a secondary slow rise (6) so that Ffj]ax remained constant for at least 10 sec. 

We would like to add to the accumulating evidence (6,7) that the fluorescence induction curve is not a well suited measurement to analyse the heterogeneity of PSII photosynthetic unit. 

Fluorescence induction was measured at 72 uE m s Photochemical (qQ) and non-photochemical (qN) fluorescence quenching were measured with 250 ms pulses (2000 uE m s ) and calculated as described in (8). Two sample-t tests were conducted at the. 05 and. 10 alpha levels and their results are reported as p-values. 

Fig 1. Comparison of Chlorophyll fluorescence induction kinetics curves of 6 wheat Cultivars (W-1...W-6) in the order of decreasing frost resistance. Each curve was plotted after accumulation and average from 10 measurements. 3 leaf sections for one measurement. 

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