An Analysis of the Mechanism of the Low-wave Phenomenon of Chlorophyll Fluorescence

The low-wave phenomenon, i.e., the transient drop of yield of modulated chlorophyll fluorescence shortly after application of a pulse of saturating light, was investigated in intact leaves of tobacco and Camellia by measuring fluorescence, CO(2) assimilation and absorption at 830 nm simultaneously. Limitations on linear electron flow, due to low electron acceptor levels that were induced by low CO(2), induced the low waves of chlorophyll fluorescence. Low-wave amplitudes obtained under different CO(2) concentrations and photon-flux densities yielded single-peak curves when plotted as functions of fluorescence parameters such as PhiPS II (quantum yield of Photosystem II) and qN (coefficient of non-photochemical quenching), suggesting that low-wave formation depends on the redox state of the electron transport chain. Low waves paralleled redox changes of P700, the reaction center of Photosystem I (PS I), and an additional electron flow through PS I was detected during the application of saturating pulses that induced low-waves. It is suggested that low waves of chlorophyll fluorescence are induced by increased non-photochemical quenching, as a result of the formation of a trans-thylakoid proton gradient due to cyclic electron flow around PS I.     

Light-induced heat production correlated with fluorescence and its quenching mechanisms

Light-induced heat produced by the non-radiative decay represents one way of de-excitation after excitation by light absorption. It was detected in vivo with cotyledons of radish seedlings (Raphanus sativus L.) by measuring the photoacoustic signal at a modulation frequency of 279 Hz. During the induction kinetic of photosynthesis the photoacoustic signal, the chlorophyll fluorescence as well as the photochemical and the non-photochemical quenching of fluorescence were simultaneously determined in order to get information about the correlation of heat production, fluorescence and its quenching mechanisms. Our results demonstrate that the changes of the photoacoustic signal can in most cases be related directly or indirectly to changes in the photochemical activity. However the kinetic of the photoacoustic signal differs from that of the fluorescence and from that of the non-photochemical quenching. This indicates that the sum of energy dissipation processes resulting in the production of light-induced heat and measured by the high-frequency photoacoustic signal must be taken into account when judging photosynthetic activity.Abbreviations LED light-emitting diode - PA photoacoustic - PAM pulse-amplitude-modulated     

Induction of photochemical and non-photochemical quenching of chlorophyll fluorescence by low concentrations of m-dinitrobenzene

Chlorophyll fluorescence quenching induced by low concentrations of m-dinitrobenzene (DNB) is investigated. In intact spinach chloroplasts DNB causes photochemical and non-photochemical quenching. The two forms of quenching are distinguished by applying the saturation pulse method with a new type of modulation fluorometer. Half-maximal photochemical quenching is observed at about 3 micromolar DNB. It is inhibited by 3-(3,4 dichlorophenyl)-1, 1-dimethylurea (DCMU) and by 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). Photochemical quenching by DNB leads to suppression of the I-P transient in a fluorescence induction curve. Upon application of saturating continuous light, the increase of fluorescence yield is separated into a photochemical and a thermal part. DNB causes suppression of only the slowest sub-component of the thermal part, in analogy to the action of Hill reagents. Simultaneous measurements of oxygen exchange rate and fluorescence reveal that a part of DNB induced quenching is accompanied by oxygen uptake. Most DNB-induced non-photochemical quenching is prevented by nigericin and, hence, can be considered energy-dependent quenching. The small component persisting in the presence of nigericin is identical to the one observed with methylviologen and other Hill reagents, likely to be due to static quenching by oxidized plastoquinone. The presented data confirm the original finding of Etienne and Lavergne (Biochim Biophys Acta 283: 268–278, 1972) that low concentrations of DNB selectively affect the thermal component of variable fluorescence. However, while these authors interpreted the quenching by a non-photochemical mechanism, the present investigation emphasizes a photochemical mechanism, in analogy to the effect of electron acceptors or mediators.Abbreviations DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1, 1-dimethylurea - DNB m-dinitrobenzene - PGA 3-phosphoglycerate - PMS phenazinemethosulphate - PS I and PS II photosystems I and II     

Rapidly reversible chlorophyll fluorescence quenching induced by pulses of supersaturating light in vivo

The saturation pulse method provides a means to distinguish between photochemical and non-photochemical quenching, based on the assumption that the former is suppressed by a saturating pulse of light (SP) and that the latter is not affected by the SP. Various types of non-photochemical quenching have been distinguished by their rates of dark relaxation in the time ranges of seconds, minutes, and hours. Here we report on a special type of non-photochemical quenching, which is rapidly induced by a pulse of high-intensity light, when PS II reaction centers are closed, and rapidly relaxes again after the pulse. This high-intensity quenching, HIQ, can be quantified by pulse-amplitude-modulation (PAM) fluorimetry (MULTI-COLOR-PAM, high sensitivity combined with high time resolution) via the quasi-instantaneous post-pulse fluorescence increase that precedes recovery of photochemical quenching in the 100–400-µs range. The HIQ amplitude increases linearly with the effective rate of quantum absorption by photosystem II, reaching about 8% of maximal fluorescence yield. It is not affected by DCMU, is stimulated by anoxic conditions, and is suppressed by energy-dependent non-photochemical quenching (NPQ). The HIQ amplitude is close to proportional to the square of maximal fluorescence yield, Fm′, induced by an SP and varied by NPQ. These properties are in line with the working hypothesis of HIQ being caused by the annihilation of singlet excited chlorophyll a by triplet excited carotenoid. Significant underestimation of maximal fluorescence yield and photosystem II quantum yield in dark-acclimated samples can be avoided by use of moderate SP intensities. In physiologically healthy illuminated samples, NPQ prevents significant lowering of effective photosystem II quantum yield by HIQ, if excessive SP intensities are avoided.

     

Fluorescence clamp: A direct measure of fluxes into and out of the antenna pool of Photosystem II

Fluorescence clamp (FC) is a method of directly measuring the fluxes out of Photosystem II antenna. This is achieved by a feed-back loop which controls the light intensity of light emitting diodes in order to keep the amplitude of modulated chlorophyll fluorescence constant, and by taking the intensity or the current fed into the light emitting diodes as a measure of the fluxes. Saturating flashes serve to distinguish between fluxes into thermal deactivation and into the photosynthetic electron transfer chain (ETC). As FC is only active in the light period of the measuring light, the background signal (induced by actinic light) is compensated by a second feed-back loop in the dark period of the measuring light. Equations are provided for the interpretation of the FC signals. This includes the quenching parameters of chlorophyll fluorescence, the flux into the electron transfer chain and the redox state of Q_A. Experiments are presented which show that traditional fluorescence (LC) and FC measurements yield the same results. However, the FC method provides a better presentation of fluxes as the scaling factor (flux/signal) is constant for all states of Photosystem II. This leads to a simpler analysis of quenching mechanisms. Examples are given which show that the co-existing quenching mechanisms with different effects on photochemical and non-photochemical fluxes can be better identified by FC rather than by LC. This revised version was published online in June 2006 with corrections to the Cover Date.     

A quantitative determination of photochemical and non-photochemical quenching during the slow phase of the chlorophyll fluorescence induction curve of bean leaves

Estimations of the changes in the reduction-oxidation state of Photosystem II electron acceptors in Phaseolus vulgaris leaves were made during the slow decline in chlorophyll fluorescence emission from the maximal level at P to the steady-state level at T. The relative contributions of photochemical and non-photochemical processes to the fluorescence quenching were determined from these data. At a low photon flux density of 100 μmol · m^?2 · s^?1, non-photochemical quenching was the major contributor to the fluorescence decline from P to T, although large charges were observed in photochemical quenching immediately after P. On increasing the light intensity 10-fold, the contribution of photochemical processes to fluorescence quenching was markedly diminished, with nearly all the P-to-T fluorescence decline being attributable to changes in non-photochemical quenching. The possible factors responsible for changes in non-photochemical quenching within the leaves are discussed.     

‘Low-waves>’ in chlorophyll fluorescence kinetics indicate deprivation of bicarbonate*

A brief reversible lowering of chlorophyll fluorescence yield (so called low-waves) immediately after application of a saturating light pulse in parallel with a short-time enhancement of the P700 oxidation level was observed in the green alga Haematococcus pluvialis. The phenomenon occurred in the steady-state time region of fluorescence induction kinetics under mild acidic conditions, and was eliminated by bicarbonate. Shortly after expression of low-waves, the photosynthetic oxygen evolution rate decreased and the non-photochemical chlorophyll fluorescence quenching component increased. The enhancement of the non-photochemical chlorophyll fluorescence quenching component was nigericin-sensitive indicating its dependence on the transthylakoid proton gradient. On the other hand, the formation of low-waves was not removed by the uncoupler. Only when bicarbonate was applied additionally, the reversible short-term decrease in fluorescence yield following each saturating light flash was abolished. Dimethyl-4-nitroso-aniline as an artificial electron acceptor of Photosystem I did not limit the brief drops in fluorescence. However, formate as a competitive inhibitor of bicarbonate binding in Photosystem II induced low-wave formation. Therefore, our results suggest that low-waves in chlorophyll fluorescence kinetics indicate deprivation of bicarbonate in the reaction centre of Photosystem II. This revised version was published online in June 2006 with corrections to the Cover Date.     

Are saturating pulses indeed saturating? Evidence for considerable PSII yield underestimation in leaves adapted to high levels of natural light

The "saturating pulse" method of in vivo Chl fluorescence measurement has been widely used by physiologists and especially ecophysiologists, as it allows a simple, rapid and non-invasive assessment of PSII function and the allocation of absorbed energy into photochemical and non-photochemical processes. It is based on the accurate determination of the so-called Fm('), i.e. the fluorescence signal emitted when a "saturating" light pulse closes all PSII centers. In this methodological investigation, we examined whether the saturating pulse intensities required to obtain maximal fluorescence yields differ between leaves of various species receiving varying actinic light irradiances. It was shown that, in leaves adapted to comparatively high (yet realistic) levels of natural irradiances, the saturating pulses usually applied are not able to close all PSII reaction centers. As a result, there is a high risk of considerable Fm(') underestimation. Accordingly, the derived values of effective PSII yields and linear electron transport rates (ETR) are also underestimated, even at the highest saturation pulse levels afforded by commercial instruments. Since the extent of underestimation increases with actinic irradiance, the ETR versus light curves are considerably distorted. The possible reasons for the apparent inability of "saturating" pulses to close all PSII centers at high actinic light and the practical implications, especially in field work, are discussed.     

濒危植物长序榆(Ulmus elongata)幼苗叶绿素荧光特性的初步研究

用Li-6400XT便携式光合作用仪对濒危植物长序榆幼苗的各叶绿素荧光参数的日变化和快速光响应曲线进行了测定。结果发现,光系统Ⅱ(PSⅡ)的实际光化学效率(ΦPSⅡ)、电子传递速率(ETR)在整个白天阶段较稳定,下午18:00显著下降。光化学淬灭(qP)先增大后减小。非光化学淬灭(NPQ)呈现出与光化学淬灭(qP)相反的变化趋势,中午最低,说明长序榆幼苗光能利用率较高。快速光曲线表明实际光化学效率(ΦPSⅡ)和光化学淬灭(qP)随着光合有效辐射(PAR)的增大而减小,电子传递速率(ETR)和非光化学淬灭(NPQ)随着光合有效辐射(PAR)的增大而增大。使用幂函数能够很好的拟合实际光化学效率(ΦPSⅡ)和电子传递速率(ETR)随光强的变化,而对数函数能较好的拟合实际光化学淬灭(qP)和非光化学淬灭(NPQ)随光强的变化。     

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.