PSI Electron Acceptors Research Articles

Studies on the O-J-I-P transient of chlorophyll fluorescence in relation to photosystem II assembly and heterogeneity in plastids of greening barley

The polyphasic variable fluorescence in saturating light (O-J-I-P transient, Strasser et al. (1995) Photochem. Photobiol. 61: 21–42) has been investigated in etiochloroplasts during the greening of etiolated leaves of Hordeum vulgare. The initial photochemical phase (O-J) due to reduction of the primary quinone acceptor Q A was found to represent a constant proportion (65–70%) of total variable fluorescence independent of greening time. The partial fluorescence quenching in the Q A-reduced state seems, therefore, to represent a basic property of PSII electron transport. The biphasic character of the slower J-I-P transient due to reduction of the plastoquinone pool developped progressively during the first hours of greening. In the same period of time the proportion and rate constant of rapid PSIIα sub-population increased, as calculated from the induction curve in the presence of DCMU. Etiochloroplasts or chloroplasts resuspended in low salt medium showed a low I level, which was restored upon readdition of 5 MM MgCl 2 and NaCl. Salts also increased the apparent proportion of PSIIα. These results suggest that the J-I and I-P phases of the induction curve are related to different rates of plastoquinone photoreduction by two distinct PSII populations. The effects of DMQ and of DCBQ on the O-J-I-P transient were also studied in (etio-)chloroplasts. In addition to the already reported quenching of the initial ( F O ) and variable fluorescence by DCBQ, a slow fluorescence increase phase was found to appear upon the addition of DCBQ but not of DMQ. The latter observations confirm that DCBQ differs from DMQ by its higher efficiency as PSII electron acceptor.

The use of photoacoustic spectroscopy in assessing leaf photosynthesis under copper stress: correlation of energy storage to photosystem II fluorescence parameters and redox change of P 700

The effects of Cu-ions on an in vivo Cu-treated leaf system of Thlaspi ochroleucum have been identified by using photoacoustic, modulated fluorescence methods and leaf absorbance. The deficiency in light harvesting capacity of PSII caused by the Cu-induced chlorophyll loss may result in the energy distribution of the two photosystems being out of balance. Copper decreased maximal fluorescence (F m, F m′) and changes at the variable fluorescence were also observed. The reduction state of the primary electron acceptor (Q A) of PSII in leaves resulted in changes due to the imbalance between the rate of Q A reduction by PSII activity and the rate of Q A − reoxidation by PSI activity. Copper treatment also resulted in a dramatic decrease of Rfd (vitality index), a moderate decrease in PES (energy storage) and a marginal decrease in F v′ F m′ values. High Cu concentrations inhibit O 2 evolution and the enhanced heat emission may be due to an inhibition of electron transfer and consequently of photochemistry. Thlaspi leaves maintained an increased capacity for PSI-driven energy storing electron flow, since measurements of 820 nm absorbance changes in saturating far-red light indicated less pronounced changes in the reaction center of PSI (P 700) activity after Cu exposure compared with that of PSII.

Superoxide contributes to the rapid inactivation of specific secondary donors of the photosystem II reaction center during photodamage of manganese-depleted photosystem II membranes.

The role of superoxide in the mechanism of photoinactivation of the secondary donors of the reaction center of photosystem II membranes depleted of Mn by extraction with NH2OH plus EDTA (NH2OH/EDTA-PSII) was assessed. EPR analyses (g = 2 region) in continuous light, optical kinetic spectrophotometric analyses of P680+ and Car+, and AT-band emission measurements were made after various durations of weak and strong light treatment of NH2OH/EDTA-PSII in the presence and absence of superoxide dismutase, or of PSII electron acceptors to suppress superoxide formation. Additionally, flash-induced variable fluorescence of chlorophyll a and the capabilities of the membranes of photooxidize Mn2+ (in the presence of H2O2) via a high-affinity site (Km approximately 180 nM) and to carry out the photoactivation of the Mn-cluster were determined. In the absence of any additions to the NH2OH/EDTA-PSII membranes which were highly depleted of Mn, weak light treatment caused rapid (t1/2 approximately 20 s) and parallel losses of (a) the approximately 10 microseconds phase of P680+ reduction, which reflects the TyrZ-->P680+ reaction, (b) the amplitude of chlorophyll a variable fluorescence, (c) the capability to accumulate the TyrZ(+)-radical in continuous light, and (d) the capability to photooxidize Mn2+/H2O2 in continuous light. As reported previously [Blubaugh et al. (1991) Biochemistry 30, 7586-7597], a dark-stable 12-G-wide featureless EPR signal centered at g = 2.004 was formed rapidly during illumination. This signal previously was tentatively identified as a Car+ radical and was suggested to contribute to the quenching of chlorophyll a variable fluorescence and to the slowing of the TyrZ-->P680+ reaction. However, we failed to detect Car+ formation by sensitive optical spectrophotometry and obtained no definable evidence for either a quencher of fluorescence other than P680+ itself or a slowing of the TyrZ-->P680+ reaction. Addition of a saturating concentration (96 units/mL) of superoxide dismutase diminished the rate of photodamage(s) by approximately 30-fold, but did not abolish it completely. Superoxide dismutase similarly suppressed strong light-induced photodamages, causing the loss of capability to photooxidize Mn2+/H2O2, to carry out photoactivation, and to generate the AT-band emission as well as TyrZ+ EPR signal. In contrast to others, we found no evidence that the initial target(s) of photodamage is (are) different in weak versus strong light treatment. The totality of the results suggests that the initial event in either weak light or strong light photodamage of NH2OH/EDTA-PSII is a decoupling of the redox activity of TyrZ from P680.(ABSTRACT TRUNCATED AT 400 WORDS)

Changes in P-700 Oxidation during the Early Stages of the Induction of Photosynthesis

Following dark adaptation, the response to irradiance of chlorophyll (Chl) fluorescence, the light-induced absorbance change around 820 nm (to measure reaction center Chl of photosystem I [PSI] P-700 oxidation), and CO2 fixation were examined in pea (Pisum sativum L.) leaves under a range of conditions. Initially, P-700 oxidation is restricted by a lack of regeneration of PSI electron acceptors, and the increase of oxidized P-700 (P-700+) that occurs during approximately the first 60 s of irradiation is largely independent of the resistance to electron flow between the two photosystems. Under these conditions, the quantum efficiency for linear electron flow is directly positively related to P-700+ accumulation, which is in contrast to the direct negative correlation that is the most frequently reported relationship between P-700+ accumulation and the quantum efficiency for linear electron flow.

Chlorophyll fluorescence in the resurrection plant Selaginella lepidophylla (Hook. & Grev.) Spring during high-light and desiccation stress, and evidence for zeaxanthin-associated photoprotection

The function of photosystem (PS)II during desiccation and exposure to high photon flux density (PFD) was investigated via analysis of chlorophyll fluorescence in the desert resurrection plant Selaginella lepidophylla (Hook. and Grev.) Spring. Exposure of hydrated, physiologically competent stems to 2000 μmol · m−2 · s−1 PFD caused significant reductions in both intrinsic fluorescence yield (FO) and photochemical efficiency of PSII (FV/FM) but recovery to pre-exposure values was rapid under low PFD. Desiccation under low PFD also affected fluorescence characteristics. Both FV/FM and photochemical fluorescence quenching remained high until about 40% relative water content and both then decreased rapidly as plants approached 0% relative water content. In contrast, the maximum fluorescence yield (FM) decreased and non-photochemical fluorescence quenching increased early during desiccation. In plants dried at high PFD, the decrease in FV/FM was accentuated and FO was reduced, however, fluorescence characteristics returned to near pre-exposure values after 24-h of rehydration and recovery at low PFD. Pretreatment of stems with dithiothreitol, an inhibitor of zeaxanthin synthesis, accelerated the decline in FV/FM and significantly increased FO relative to controls at 925 μmol · m−2 · s−1 PFD, and the differences persisted over a 3-h low-PFD recovery period. Pretreatment with dithiothreitol also significantly decreased non-photochemical fluorescence quenching, increased the reduction state of QA, the primary electron acceptor of PSII, and prevented the synthesis of zeaxanthin relative to controls when stems were exposed to PFDs in excess of 250 μmol · m−2 · s−1. These results indicate that a zeaxanthin-associated mechanism of photoprotection exists in this desert pteridophyte that may help to prevent photoinhibitory damage in the fully hydrated state and which may play an additional role in protecting PSII as thylakoid membranes undergo water loss.

Discovery and Optimization of a PSI Electron‐Accepting 1,2,4‐Benzotriazine Herbicide

AbstractA random source PSI electron‐accepting herbicide lead, ethyl 1,2,4‐benzotriazine‐3‐acetate 1, was optimized using molecular probing, bioisosteric replacement, Free‐Wilson analysis, Sequential Simplex Optimization, and ultimately linear regression analysis. Some variables in the initial regression equations were found to be cross correlated, representing the same factors as revealed by factor analysis. The technique of prefiltering potential variables for linear regression analysis utilizing factor analysis is exemplified. For the set of 1 analogs, eleven independent factors were identified which were then submitted to linear regression analysis. The resulting equations are statistically more secure, all terms being independent. These equations are discussed in the context of known SAR for herbicidal PSI electron acceptors, especially the bipyridiniums.

Energy Storage of Linear and Cyclic Electron Flows in Photosynthesis

The energy storage of photosynthesis in the green alga Chlorella vulgaris was determined by pulsed, time-resolved photoacoustics. The energy storage of the linear electron transfer process in photosynthesis, of cyclic photosystem (PS) I, and possibly of PSII was determined by selection of excitation wavelength and of flash interval. At 695 nm excitation, a rather large cyclic PSI energy storage of 0.68 +/- 0.04 eV/quantum of energy at 8 ms after a 1-mus flash was obtained. This energy remained the same at flash intervals of 0.35 to 60 s and was independent of the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. We tentatively assign this energy to the ferredoxin-NADP-reductase-ferredoxin and oxidized cytochrome b(6)/f complexes. An efficient distribution of energy between cyclic and linear systems is obtained with the simple assumption that the turnover time of the cyclic system is slower than that of the linear system. The energy storage of linear electron flow was determined by 655 nm excitation of Chlorella with a short flash interval of 0.35 s per flash. It was calculated to be 0.50 +/- 0.03 eV/hv, close to that expected for oxygen and NADPH formation. The energy storage of PSII is determined by excitation of Chlorella at 655 nm with a long flash interval of 60 s per flash. It was calculated to be 1.07 +/- 0.05 eV/hv, consistent with the energy storage being in S-states and the secondary electron acceptor of PSII with a calculated redox energy of 1.03 eV/hv. In the presence of 1 mum 3-(3,4-dichlorophenyl)-1,1-dimethylurea, the calculated energy storage in PSII is still significant, 0.53 +/- 0.04 eV/hv. This probably indicates a significant cyclic electron flow around PSII. These cyclic flows may contribute considerably to energy storage in photosynthesis.

Analysis of dark-relaxation kinetics of variable fluorescence in intact leaves

The dark-relaxation kinetics of variable fluorescence, Fv, in intact green leaves of Pisum stativum L. and Dolichos lablab L. were analyzed using modulated fluorometers. Fast (t1/2 = 1 s) and slow (t1/2 = 7-8 s) phases in fv dark-decay kinetics were observed; the rate and the relative contribution of each phase in total relaxation depended upon the fluence rate of the actinic light and the point in the induction curve at which the actinic light was switched off. The rate of the slow phase was accelerated markedly by illumination with far-red light; the slow phase was abolished by methyl viologen. The halftime of the fast phase of Fv dark decay decreased from 250 ms in dark-adapted leaves to 12-15 ms upon adaptation to red light which is absorbed by PSII. The analysis of the effect of far-red light, which is absorbed mainly by PSI, on Fv dark decay indicates that the slow phase develops when a fraction of QA (-) (the primary stable electron acceptor of PSII) cannot transfer electrons to PSI because of limitation on the availability of P700(+) (the primary electron donor of PSI). After prolonged illumination of dark-adapted leaves in red (PSII-absorbed) light, a transient. Fv rise appears which is prevented by far-red (PSI-absorbed) light. This transient fv rise reflects the accumulation of QA (-) in the dark. The observation of this transient Fv rise even in the presence of the uncoupler carbonylcyanide m-chlorophenyl hydrazone (CCCP) indicates that a mechanism other than ATP-driven back-transfer of electrons to QA may be responsible for the phenomenon. It is suggested that the fast phase in Fv dark-decay kinetics represents the reoxidation of QA (-) by the electron-transport chain to PSI, whereas the slow phase is likely to be related to the interaction of QA (-) with the donor side of PSII.

A SPECTROPHOTOMETRIC METHOD FOR CHLOROPLAST ATP SYNTHETASE ASSAY

In the present work an assay method of chloroplast adenosine triphosphate (ATP) synthetase has been studied in which the production of ATP from thylakoids was monitored spectrophotometrically by the reduction of NADP+via hexokinase and glucose-6-phosphate dehydrogenase reactions. The interference caused by photosynthetic NADPH production was completely eliminated by use of methyl viologen, a PSI electron acceptor. Under the assay conditions established in this study, the rate of ATP synthesis was directly proportional to the amount of chloroplast ATP synthetase. The method appears reliable, convenient and considerably sensitive. In addition, this technique provides a continuous measurement of ATP production from thylakoids with a limiting adenosine diphosphate concentration.

The Mechanism of Herbicide Resistance in Tobacco Cells with a New Mutation in the QB Protein

A new mutant of the psbA gene conferring resistance to 2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine (atrazine) was obtained by selection of photomixotrophic tobacco (Nicotiana tabacum cv Samsun NN) cells. The 264th codon AGT (serine) in the wild psbA gene was changed to ACT (threonine) in these mutant tobacco cells. All other higher plants resistant to atrazine exhibit a change to GGT (glycine) in this codon. Measurements of Hill reaction activity and chlorophyll fluorescence showed that the threonine 264-containing plastoquinone serving as secondary stable electron acceptor of PSII (Q(B) protein) had not only strong resistance to triazine-type herbicides but also moderate resistance to substituted urea-type herbicides. Threonine-type Q(B) protein showed especially strong resistance to methoxylamino derivatives of the substituted urea herbicides. The projected secondary structures of the mutant Q(B) proteins indicate that the cross-resistance of threonine 264 Q(B) protein to triazine and urea herbicides is mainly due to a conformational change of the binding site for the herbicides. However, the glycine 264 Q(B) protein is resistant to only triazine herbicides because of the absence of an hydroxyl group and not because of a conformational change.

Artificial inhibitory effects of sulfite on photosystem II activity measured by oxygen evolution in chloroplasts

In this report we demonstrate sulfite interaction with oxygen and PSII electron acceptors (ferricyanide and para-benzoquinone) during measurement of oxygen evolution in chloroplasts. Redox potentials of oxygen, ferricyanide and para-benzoquinone allow them to compete for sulfite. Without taking this into account, sulfite inhibition of oxygen evolution can be overestimated, since sulfite consumes oxygen and reduces ferricyanide or para-benzoquinone during the measurement. In order to correctly measure the rate of oxygen evolution in chloroplasts, it is necessary to avoid presence of sulfite during the measurement. After overcoming the artifact, mentioned above, we confirm the sulfite inhibition of oxygen evolution in chloroplasts but at a lesser extent than earlier reported. This, however, is a pretreatment effect.

Chlorophyll fluorescencein vivo as an indicator of water stress in potato leaves

Potato plants, cv. Desiree, were exposed to a -0.3 MPa polyethylene glycol-induced water stress for 24 h in a hydroponic system. Leaves with a Leaf Plastochron Index of 3 to 3.99, 4 to 4.99 and 6 to 6.99 were measured for total, osmotic and pressure potential, leaf conductance andin vivo chlorophyll fluorescence transients Fo, Fp and FT. Leaf conductance and Fo significantly declined with leaf age in unstressed leaves. Water potential components and leaf conductance were significantly lower after 4 h of water stress whereas all three chlorophyll fluorescence transients measured after 24 h were significantly higher. The increase in fluorescence transients suggested that PSII electron acceptors were reduced faster than PSI and the Calvin cycle could oxidize them.In vivo chlorophyll fluorescence provides a greater understanding of the physiological effects of water stress but it requires additional experimentation to make it useful in screening for water stress tolerance.

Heterogeneity in chloroplast photosystem II

Photosystem-two (PSII) in the chloroplasts of higher plants and green algae is not homogeneous. A review of PSII heterogeneity is presented and a model is proposed which is consistent with much of the data presented in the literature. It is proposed that the non-quinone electron acceptor of PSII is preferentially associated with the sub-population of PSII known as PSIIß.