Biophysical Methods of Ecological Monitoring. Photosynthetic characteristics of tree plants growing in Moscow city

In this work, we studied the influence of ecological factors (distance from thoroughfares) on photosynthetic characteristics of leaves of four tree species growing in Moscow city. Photosynthetic activity of leaves was assayed by instrumental methods of probing the functional state of the photosynthetic apparatus, using electron paramagnetic resonance for measuring the kinetics of photooxidation of P₇₀₀ centers, thermoluminescence, and slow induction of chlorophyll fluorescence. It has been shown that the kinetic parameters of the induction curves, as determined from the kinetics of P₇₀₀ photooxidation and slow fluorescence induction in dark-adapted leaves, are sensitive to variations of plant growth conditions. These parameters can be used as informative characteristics in ecological monitoring.     

The ontogenetic approach to chlorophyll fluorescence studies of plant photosynthetic apparatus under stressful conditions

Based on the analysis of reasons limiting the application of the method of chlorophyll fluorescence induction for estimating the state of the leaf photosynthetic apparatus under prolonged stress, the necessity of the ontogenetic approach consisting in a more exact determination of leaf age was substantiated. A comparison of the calendar and ontogenetic ways of determination of age of cucumber leaves under controlled conditions revealed essential distinctions in the estimation of plant leaf photosynthetic apparatus by the method of chlorophyll fluorescence induction for two variants distinguishing by the cultivation light regime ("white", 400-700 nm, and "red", 600-700 nm). It was shown that, in the case of prolonged effect of the stress factor on the plant, the unambiguity of the interpretation of chlorophyll fluorescence induction parameters in the estimation of the state of their photosynthetic apparatus depends essentially on the choice of the ontogenetic period of leaves of plants being compared and the accuracy of determination of leaf age.     

Luminescent indicators in various parts of wheat leaves in ontogenesis

A comparative study of slow fluorescence induction, fluorescence spectra, thermoluminescence, photosynthetic activity and chlorophyll content in ontogenesis of wheat seedlings (Triticum aestivum L, cv. Yubileinayd) grown in the laboratory conditions was carried out. It was shown that (FM-FT)/FT values of slow fluorescence induction increased with the age of leaf part, reached a maximum (for 2-week-old seedlings), and then decreased. Changes in (FM-FT)/FT positively correlated with the changes in photosynthetic activity per chlorophyll unit (delta O2/(delta t.chlorophyll)); the coefficient of correlation r = 0.84, p > 0.999. The F685/F730 ratio of the intensities of fluorescence spectrum decreased with the increase in chlorophyll content. The relative light sum SA/S(tot) of band A of thermoluminescence changed in the same direction as delta O2/(delta t-chlorophyll), the relative light sum (SC/S(tot) of band C of thermoluminescence decreased during leaf maturation. The regularities revealed in the experiments are discussed in terms of changes in the structural and functional organization of the photosynthetic apparatus known from the literature.     

Manganese limitation induces changes in the activity and in the organization of photosynthetic complexes in the cyanobacterium Synechocystis sp. strain PCC 6803

Manganese (Mn) ions are essential for oxygen evolution activity in photoautotrophs. In this paper, we demonstrate the dynamic response of the photosynthetic apparatus to changes in Mn bioavailability in cyanobacteria. Cultures of the cyanobacterium Synechocystis PCC 6803 could grow on Mn concentrations as low as 100 nm without any observable effect on their physiology. Below this threshold, a decline in the photochemical activity of photosystem II (PSII) occurred, as evident by lower oxygen evolution rates, lower maximal photosynthetic yield of PSII values, and faster Q(A) reoxidation rates. In 77 K chlorophyll fluorescence spectroscopy, a peak at 682 nm was observed. After ruling out the contribution of phycobilisome and iron stress-induced IsiA proteins, this band was attributed to the accumulation of partially assembled PSII. Surprisingly, the increase in the 682-nm peak was paralleled by a decrease in the 720-nm peak, dominated by PSI fluorescence. The effect on PSI was confirmed by measurements of the P(700) photochemical activity. The loss of activity was the result of two processes: loss of PSI core proteins and changes in the organization of PSI complexes. Blue native-polyacrylamide gel electrophoresis analysis revealed a Mn limitation-dependent dissociation of PSI trimers into monomers. The sensitive range for changes in the organization of the photosynthetic apparatus overlaps with the range of Mn concentrations measured in natural environments. We suggest that the ability to manipulate PSI content and organization allows cyanobacteria to balance electron transport rates between the photosystems. At naturally occurring Mn concentrations, such a mechanism will provide important protection against light-induced damage.     

Oxygen as an alternative electron acceptor in the photosynthetic electron transport chain of C3 plants

This study deals with effects of oxygen on the kinetics of P(700) photoinduced redox transitions and on induction transients of chlorophyll fluorescence in leaves of C(3) plants Hibiscus rosa-sinensis and Vicia faba. It is shown that the removal of oxygen from the leaf environment has a conspicuous effect on photosynthetic electron transport. Under anaerobic conditions, the concentration of oxidized P700 centers in continuous white light was substantially lower than under aerobic conditions. The deficiency of oxygen released non-photochemical quenching of chlorophyll fluorescence, thus indicating a decrease in the trans-thylakoid pH gradient (DeltapH). Quantitative analysis of experimental data within the framework of an original mathematical model has shown that the steady-state electron flux toward oxygen in Chinese hibiscus leaves makes up to approximately 40% of the total electron flow passing through photosystem 1 (PS1). The decrease in P700+ content under anaerobic conditions can be due to two causes: i) the retardation of electron outflow from PS1, and ii) the release of photosynthetic control (acceleration of electron flow from PS2 to P700+) owing to lower acidification of the intra-thylakoid space. At the same time, cyclic electron transport around PS1 was not stimulated in the oxygen-free medium, although such stimulation seemed likely in view of possible rearrangement of electron flows on the acceptor side of PS1. This conclusion stems from observations that the rates of P700+ reduction in DCMU-poisoned samples, both under aerobic and anaerobic conditions, were negligibly small compared to rates of electron flow from PS2 toward P700+ in untreated samples.     

Discrete character of the development of the photosynthetic apparatus in greening barley leaves

Biogenesis of the photosynthetic apparatus in greening etiolated leaves of barley (Hordeum vulgare L) was investigated by an approach permitting investigation of this process under conditions that minimize differences in plastid development. Distributions of barley leaves greening for 24 h as to chlorophyll content and of chloroplast grana as to number of thylakoids were shown to be of a multimodal character. The shape of time-course curves of chlorophyll accumulation in local sites of greening etiolated leaves was of a stepped or (at the end of greening) undulated character. The stepwise accumulation of chlorophyll was accompanied by wave-like changes in chlorophyll b/a ratio, intensity of low-temperature chlorophyll fluorescence and photosynthetic activity with minima at the time points of transition to accelerated chlorophyll accumulation. It is assumed that (1) development of the photosynthetic apparatus in local sites of greening etiolated leaves occurs stepwise, from one steady level to another, but not as gradually as is generally accepted, and (2) every separate step in development of the photosynthetic apparatus seems to begin with formation of photosystem cores and to end with the synthesis of light-harvesting complexes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of choline chloride on photosynthetic oxygen release and variable and delayed fluorescence of chlorophyll in Chlamydomonas reinhardtii cells

The influence of the growth retardant chlorocholine chloride on the rate of photosynthetic oxygen evolution and the induction of chlorophyll fluorescence in unicellular green algae Chlamydomonas was studied depending on concentration and the time of cell growth. It was shown that low concentrations chlorocholine chloride (0.02 g/l) affected insignificantly the photosynthesis and chlorophyll fluorescence. The growth of the culture in the presence of higher chlorocholine chloride concentrations (0.2 and 2 g/l) led to a significant reduction in the rate of oxygen production, and photoinduced changes in chlorophyll fluorescence yield. Young cells were more sensitive to chlorocholine chloride than old cells.     

Heat sensitivity and thermal adaptation of photosynthesis in liverwort thalli

Summary The effect of high temperatures on the photosynthetic apparatus of Preissia quadrata (Scop.) Nees, Conocephalum conicum (L.) Dum. and Marchantia polymorpha L. were investigated. changes in the activities of various photosynthetic reactions were followed by measuring light-dependent oxygen evolution, chlorophyll a fluorescence and light-induced absorbance changes at 518 nm.Mild heat treatment of the thalli led to reversible depression of photosynthesis; the period necessary for complete recovery depended on the extent of heat damage. Irreversible inactivation of photosynthesis after more severe heat stress was caused by damage of photosystem II. On principle, the pattern of reversible and irreversible heat inactivation of photosynthetic reactions in liverwort thalli resembles that observed in leaves of higher plants. However, in contrast to a number of Spermatophyta, exposure of liverwort thalli to high sublethal temperatures did not result in a significant increase in the heat stability of the photosynthetic apparatus indicating that the heat hardening capacity of hygrophytic hepatics is extremely low.     

Effects of low temperature acclimation upon photosynthetic induction in barley primary leaves

Oxygen evolution and chlorophyll fluorescence were measured in cold-hardened and unhardened leaves of barley ( Hordeum vulgare L. cv. Asa) during the induction period of photosynthesis. The lag phase of light-saturated photosynthesis was increased and steady-state rates of photosynthesis were higher in cold-hardened than in unhardened barley leaves. Fluorescence was quenched more rapidly during the first minutes of induction in hardened than unhardened leaves, largely because of greater energy-dependent quenching (q_E). Also, slow fluorescence transients through the M peak were delayed and less pronounced in cold-hardened than in unhardened leaves. Based upon the combined fluorescence and oxygen evolution data it was concluded that cold-hardening delayed light activation of the energy consuming carbon reduction cycle, thereby delaying the use of ATP and NADPH formed in the light reaction. Measurements of oxygen evolution and fluorescence kinetics during photosynthetic induction under oxygenic and anoxygenic conditions suggest that oxygen photoreduction is important for additional ATP generation during both the onset of photosynthetic carbon assimilation and during steady-state photosynthesis.     

Evaluation of chlorophyll fluorescence as a probe for drought stress in willow leaves

The effect of drought on the photosynthetic apparatus of leaves of Salix sp. was studied by measurements of the induction of chlorophyll fluorescence and the capacity for O₂ evolution. Using a multivariate analysis, a model was developed that could predict the degree of drought stress from the data of fluorescence kinetics. Even mild drought stress was detected with high precision; this was not always possible when the photosynthetic capacity was measured. The most clear discrimination between control and drought-stressed leaves was obtained if fluorescence induction was measured at high rather than normal CO₂ levels, and at low rather than high light levels. All information provided by fluorescence pertaining to drought was contained within the slow phase of the induction curve. It is suggested that rapid dehydration is different from drought at the mechanistic level as judged by the fluorescence characteristics.     

Effect of inorganic phosphate on photosynthesis in bean leaves

It was shown that raising pod seedlings by the hydroponics method on KH2PO4 solutions at concentrations between 10(-7) and 10(-5) M leads to an increase in the rate of oxygen release (delta O2/delta t), with the chlorophyll content in leaves being unchanged. The values of the parameters FM/FT of slow fluorescence induction and B/A of photoinduced changes in ESR1 signals from pod leaves correlate with the delta O2/delta t value.     

Photochemical activities and organization of photosynthetic apparatus of C3 and C4 plants grown under different light intensities

Changes in the photochemical activities, influenced by variation in the growth light intensity, were followed in typical C₃ (Phaseolus, Ipomoea) and C₄ (Amaranthus, Sorghum) plants. Progressive decrease in the growth light intensity accelerated the O-P fluorescence induction in whole leaves. Such acceleration of the fluorescence kinetics was found to be not due to enhanced photosystem II activity but possibly a result of reduced rate of electron flow between the two photosystems. This is supported by 4 lines of evidence: (1) by the Hill activity determined in the presence of electron acceptors functioning before and after plastoquinone; (2) the photosynthetic unit size determined after flash excitation showing variations that were apparently too small to account for the changes observed fluorescence induction; (3) modification of the kinetics of secondrange light-induced absorbance changes at 520 nm; and (4) absence of significant changes in the ratio of P₇₀₀/total chlorophyll ratio. The P₇₀₀/cytochrome f ratio, however, increased from the usual 1–1.5 to 3–4 in plants grown under 9% sunlight. Increase in the P₇₀₀/cytochrome f ratio was found to be due to a decrease in the cytochrome f/chlorophyll ratio, and this was due to perhaps to a simultaneous increase in chlorophyll and decrease in cytochrome content.     

Photochemical activities and organization of photosynthetic apparatus of C3 and C4 plants grown under different light intensities

Changes in the photochemical activities, influenced by variation in the growth light intensity, were followed in typical C₃ (Phaseolus, Ipomoea) and C₄ (Amaranthus, Sorghum) plants. Progressive decrease in the growth light intensity accelerated the O-P fluorescence induction in whole leaves. Such acceleration of the fluorescence kinetics was found to be not due to enhanced photosystem II activity but possibly a result of reduced rate of electron flow between the two photosystems. This is supported by 4 lines of evidence: (1) by the Hill activity determined in the presence of electron acceptors functioning before and after plastoquinone; (2) the photosynthetic unit size determined after flash excitation showing variations that were apparently too small to account for the changes observed fluorescence induction; (3) modification of the kinetics of second-range light-induced absorbance changes at 520 nm; and (4) absence of significant changes in the ratio of P₇₀₀/total chlorophyll ratio. The P₇₀₀/cytochrome f ratio, however, increased from the usual 1–1.5 to 3–4 in plants grown under 9% sunlight. Increase in the P₇₀₀/cytochrome f ratio was found to be due to a decrease in the cytochrome f/chlorophyll ratio, and this was due to perhaps to a simultaneous increase in chlorophyll and decrease in cytochrome content.     

Acclimation of shade-tolerant and light-resistant <Emphasis Type="Italic">Tradescantia</Emphasis> species to growth light: chlorophyll <Emphasis Type="Italic">a</Emphasis> fluorescence,electron transport,and xanthophyll content

In this study, we have compared the photosynthetic characteristics of two contrasting species of Tradescantia plants, T. fluminensis (shade-tolerant species), and T. sillamontana (light-resistant species), grown under the low light (LL, 50–125 µmol photons m^?2 s^?1) or high light (HL, 875–1000 µmol photons m^?2 s^?1) conditions during their entire growth period. For monitoring the functional state of photosynthetic apparatus (PSA), we measured chlorophyll (Chl) a emission fluorescence spectra and kinetics of light-induced changes in the heights of fluorescence peaks at 685 and 740 nm (F ₆₈₅ and F ₇₄₀). We also compared the light-induced oxidation of P₇₀₀ and assayed the composition of carotenoids in Tradescantia leaves grown under the LL and HL conditions. The analyses of slow induction of Chl a fluorescence (SIF) uncovered different traits in the LL- and HL-grown plants of ecologically contrasting Tradescantia species, which may have potential ecophysiological significance with respect to their tolerance to HL stress. The fluorometry and EPR studies of induction events in chloroplasts in situ demonstrated that acclimation of both Tradescantia species to HL conditions promoted faster responses of their PSA as compared to LL-grown plants. Acclimation of both species to HL also caused marked changes in the leaf anatomy and carotenoid composition (an increase in Violaxanthin?+?Antheraxantin?+?Zeaxanthin and Lutein pools), suggesting enhanced photoprotective capacity of the carotenoids in the plants grown in nature under high irradiance. Collectively, the results of the present work suggest that the mechanisms of long-term PSA photoprotection in Tradescantia are based predominantly on the light-induced remodeling of pigment-protein complexes in chloroplasts.     

Regulation of electron transport in C(3) plant chloroplasts in situ and in silico: short-term effects of atmospheric CO(2) and O(2)

In this work, we have investigated the effects of atmospheric CO(2) and O(2) on induction events in Hibiscus rosa-sinensis leaves. These effects manifest themselves as multiphase kinetics of P(700) redox transitions and non-monotonous changes in chlorophyll fluorescence. Depletion of CO(2) and O(2) in air causes a decrease in linear electron flux (LEF) and dramatic lowering of P(700)(+) level. This is explained by the impediment to electron efflux from photosystem 1 (PS1) at low acceptor capacity. With the release of the acceptor deficit, the rate of LEF significantly increases. We have found that oxygen promotes the outflow of electrons from PS1, providing the rise of P(700)(+) level. The effect of oxygen as an alternative electron acceptor becomes apparent at low and ambient concentrations of atmospheric CO(2) < or = 0.06-0.07%). A decrease in LEF at low CO(2) is accompanied by a significant (about 3-fold) rise of non-photochemical quenching (NPQ) of chlorophyll fluorescence. Such an increase in NPQ can be explained by more significant acidification of the thylakoid lumen. This occurs due to lessening the proton flux through the ATP synthases caused by a decrease in the ATP consumption in the Bassham-Benson-Calvin (BBC) cycle. pH-dependent mechanisms of electron transport control have been described within the frames of our mathematical model. The model describes the reciprocal changes in LEF and NPQ and predicts the redistribution of electron fluxes on the acceptor side of PS1. In particular, the contribution of cyclic electron flow around PS1 (CEF1) and water-water cycle gradually decays during the induction phase. This result is consistent with experimental data indicating that under the steady-state conditions the contribution of CEF1 to photosynthetic electron transport in Hibiscus rosa-sinensis is insignificant (< or = 10%).     

Early effect of sodium pentachlorophenate on photosynthetic activity of the alga <Emphasis Type="Italic">Chlorella pyrenoidosa</Emphasis> Chick. S-39

Early effects of 0.0001–10 mM sodium pentachlorophenate (PCP-Na) on the green alga Chlorella pyrenoidosa Chick. S-39 involves a rapid (within 1–2 min) decrease in the light-induced oxygen evolution by algal cells. The suppressed relative yield of variable chlorophyll fluorescence in C. pyrenoidosa in the presence of high PCP-Na concentrations and its dynamics provide evidence for rapid inactivation of photosystem 2, which is not observed at low concentrations of the toxicant. An analysis of the induction curve of delayed chlorophyll fluorescence in algal cells suggests that PCP-Na at low concentrations disturbs the coupling of electron transport and phosphorylation, whereas at high concentrations it inhibits electron transport and decreases the energy potential of photosynthetic membranes. The early toxic effect of PCP-Na is responsible for subsequent impairment of C. pyrenoidosa productivity.     

外源亚精胺对Ca(NO3)2胁迫下番茄幼苗光合特性和抗氧化酶活性的影响

以耐盐性较弱的番茄品种上海‘合作903’幼苗为试验材料,采用营养液栽培方法,研究了叶面喷施1mmol.L-1亚精胺(Spd)对75mmol.L-1 Ca(NO32)胁迫下番茄幼苗生长、叶绿素含量、光合及荧光参数、叶片抗氧化酶活性的影响,以探讨外源亚精胺缓解Ca(NO3)2胁迫伤害的机制。结果显示:Ca(NO3)2胁迫能够显著抑制番茄幼苗的生长;与Ca(NO32)胁迫处理相比,叶面喷施外源Spd 9d后,受胁迫番茄幼苗的株高、茎粗、干重、鲜重分别显著增加70.9%、15.8%、43.4%、41.4%;叶绿素a、b的含量分别提高17.7%、13.8%;净光合速率(Pn)、气孔导度(Gs)、蒸腾速率(Tr)、光合电子传递效率(rETR)和光化学猝灭系数(qP)分别升高6.6%、18.0%、31.0%、4.9%、5.0%,而胞间二氧化碳浓度(Ci)、非光化学猝灭系数(qN)分别降低21.5%、8.1%;超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性分别增加10.7%和37.5%,而丙二醛(MDA)含量和超氧阴离子(O2.-)产生速率分别显著下降34.5%、17.1%。研究表明,外源Spd通过提高番茄幼苗抗氧化酶活性来有效清除体内活性氧,维持光合机构的稳定性,提高其光合速率,从而缓解Ca(NO3)2胁迫对番茄幼苗的伤害。     

Temperature-dependent adjustment of the thermal stability of photosystem II in vivo: possible involvement of xanthophyll-cycle pigments

Moderately elevated temperatures induce a rapid increase in the heat and light resistance of photosystem II (PSII) in higher-plant leaves. This phenomenon was studied in intact potato leaves exposed to 35 °C for 2 h, using chlorophyll fluorometry, kinetic and difference spectrophotometry and photoacoustics. The 35 °C treatment was observed to cause energetic uncoupling between carotenoids and chlorophylls: (i) the steady-state chlorophyll fluorescence emission excited by a blue light beam (490 nm) was noticeably reduced as compared to fluorescence elicited by orange light (590 nm) and (ii) the quantum yield for photosynthetic oxygen evolution in blue light (400–500 nm) was preferentially reduced relative to the quantum yield measured in red light (590–710 nm). Analysis of the chlorophyll-fluorescence and light-absorption characteristics of the heated leaves showed numerous analogies with the fluorescence and absorption changes associated with the light-induced xanthophyll cycle activity, indicating that the carotenoid species involved in the heat-induced pigment uncoupling could be the xanthophyll violaxanthin. More precisely, the 35 °C treatment was observed to accelerate and amplify the non-photochemical quenching of chlorophyll fluorescence (in both moderate red light and strong white light) and to cause an increase in leaf absorbance in the blue-green spectral region near 520 nm, as do strong light treatments which induce the massive conversion of violaxanthin to zeaxanthin. Interestingly, short exposure of potato leaves to strong light also provoked a significant increase in the stability of PSII to heat stress. It was also observed that photosynthetic electron transport was considerably more inhibited by chilling temperatures in 35 °C-treated leaves than in untreated leaves. Further, pre-exposure of potato leaves to 35 °C markedly increased the amplitude and the rate of light-induced changes in leaf absorbance at 505 nm (indicative of xanthophyll cycle activity), suggesting the possibility that moderately elevated temperature increased the accessibility of violaxanthin to the membrane-located de-epoxidase. This was supported by the quantitative analysis of the xanthophyll-cycle pigments before and after the 35 °C treatment, showing light-independent accumulation of zeaxanthin during mild heat stress. Based on these results, we propose that the rapid adjustment of the heat resistance of PSII may involve a modification of the interaction between violaxanthin and the light-harvesting complexes of PSII. As a consequence, the thermoresistance of PSII could be enhanced either directly through a conformational change of PSII or indirectly via a carotenoid-dependent modulation of membrane lipid fluidity.Abbreviations and Symbols Fo and Fm initial and maximal level of chlorophyll fluorescence, respectively - Fv = Fm — Fo variable chlorophyll fluorescence - LHC(II) light-harvesting chlorophylla/b-protein complexes (of PSII) - photoacoustically measured quantum yield of photosynthetic oxygen evolution (in relative values) - _P fluorimetrically measured quantum yield of PSII photochemistry in the light - PFD photon flux density - q_E pH dependent quenching of chlorophyll fluorescence We thank Dr. J-L Montillet (CEA-Cadarache) for the use of his HPLC apparatus and Professor Y. Lemoine (University of Lille, France) for technical advice on HPLC.     

Induction kinetics of photosystem I-activated P700 oxidation in plant leaves and their dependence on pre-energization

Absorbance changes ΔA ₈₁₀ were measured in pea (Pisum sativum L., cv. Premium) leaves to track redox transients of chlorophyll P700 during and after irradiation with far red (FR) light under various preillumination conditions in the absence and presence of inhibitors and protonophorous uncoupler of photosynthetic electron transport. It was shown that cyclic electron transport (CET) in chloroplasts of pea leaves operates at its highest rate after preillumination of leaves with white light and is strongly suppressed after preillumination with FR light. The FR light-induced suppression was partly released during prolonged dark adaptation. Upon FR illumination of dark-adapted leaves, the induction of CET was observed, during which CET activity increased to the peak from the low level and then decreased gradually. The kinetics of P700 oxidation induced by FR light of various intensities in leaves preilluminated with white light were fit to empirical sigmoid curves containing two variables. In leaves treated with a protonophore FCCP, the amplitude of FR light-induced changes ΔA ₈₁₀ was strongly suppressed, indicating that the rate of CET is controlled by the pH gradient across the thylakoid membrane.     

Effect of CO<Subscript>2</Subscript> supply on formation of reactive oxygen species in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Light-induced generation of reactive oxygen species (ROS) in 2-week-old leaves of Arabidopsis thaliana was studied by means of the ROS-sensitive dyes nitroblue tetrazolium (NBT) and 5-(and-6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate (DCF-DA). Superposition of pictures of chlorophyll fluorescence and DCF fluorescence indicated that the origin of ROS was in the chloroplasts. Experiments were done with zero, 0.1, or 10 mM NaHCO3 in the infiltration medium. Energy quenching in photosystem II was higher under low CO2 concentrations as measured by chlorophyll fluorescence. DCF fluorescence showed that CO2 deficiency led to an increase of ROS generation. In contrast, the photosystem II inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea reduced the light-induced increase of DCF fluorescence. This indicates that ROS production does not primarily result from over-reduction of photosystem II as caused by impeding electron flow in the electron transfer chain. More likely, it is an effect of diverting electron flux normally aimed at carboxylation in the Calvin cycle to other sinks more prone to the generation of toxic radicals. There was no significant effect of salicyl hydroxamate (a blocker of the alternative oxidase), showing that the mitochondrial electron transfer chain seems to play a minor role as already indicated by the superposition of chlorophyll and DCF fluorescence.