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A set of expressions is derived which quantifies the chlorophyll fluorescence yield in terms of rate constants of primary light reactions of PSII, the fraction of open and semi-open RCs and of the electric field sensed by the RC in the thylakoid membrane. The decay kinetics of the chlorophyll fluorescence yield after a single turnover excitation in the presence of DCMU show at least two components, one reversible within approx. 1 s and one with a dark reversion lasting more than 30 s. The latter is attributed to photochemical quenching; the fast component is interpreted to be associated at least partially with photo-electrochemical control. It will be illustrated that (i) the sub-maximal fluorescence yield in single turnover excitation is associated with semi-closure of RCs, (ii) the trapping efficiency of semi-closed centers is less than 50% of that of open centers and (iii) the fluorescence yield of antennas with semi-closed RCs has the highest sensitivity to changes in strength of photo-electric fields.

Exposure to ultraviolet-B radiation (UVB) radiation affects plants in multiple ways, including effects on the photosynthetic apparatus. The carbon dioxide reduction reactions are affected as well as the light reactions, especially those of photosystem II. In the literature several UVB chromophores are suggested, including redox-active tyrosines, plastosemiquinones, and the manganese cluster of the water-splitting complex. We measured the damage by UVB radiation given together with a small component of photosynthetically active radiation (PAR) in a triazine-resistant biotype and a wild-type of Chenopodium album. The damage was estimated by chlorophyll fluorescence measurements and was larger in the resistant plants. We were able to show that the concentration of the semireduced primary quinone electron acceptor of photosystem II is higher in the resistant plants, under all radiation conditions tested. This finding and additional results support the hypothesis that plastosemiquinones are photosensitizers for UVB radiation; absorption of UVB light by these quinones initiates reactions leading to damage to photosystem II.

Natural variation for photosynthetic traits was studied by determining chlorophyll fluorescence parameters in a collection of Arabidopsis accessions. This screen revealed only one single accession (Ely), exhibiting photosynthetic characteristics markedly different from all others, while a few lines showed small but significant variation. Detailed genetic and physiological analyses showed reduced fitness for Ely compared with the standard laboratory strain Ler for various growth parameters. At low temperature (15 degrees C), Ely had a higher electron transport rate than Ler, indicating increased photosystem II efficiency under this condition, while at high temperature (30 degrees C) the opposite was observed. Ely had a high sensitivity to UV-B radiation compared with Ler and was atrazine resistant. This atrazine-resistance and related chlorophyll fluorescence traits were maternally inherited, pointing towards chloroplast-located gene(s). Definite proof that Ely is atrazine-resistant was obtained by sequencing the psbA gene, encoding the D1 protein of photosystem II, revealing a point mutation causing the same amino acid change as found in other atrazine-resistant species. Additional nuclear encoded genetic variation was also present, as was concluded from the small but significant differences in phenotype between Ely and its reciprocal crosses with Ler. It was concluded that the photosynthetic yield is highly conserved and that only severe selection pressure results in marked variations in photosynthetic performance.

A series of replacement experiments of [14C]-triazines, [14C]-atrazine and [7-14C]-2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine, bound to thylakoids isolated from wild-type and atrazine-resistant Chenopodium album (lambsquarters) were conducted. Replacement experiments of [14C]-triazines bound to wild-type Chenopodium thylakoids with non-labeled atrazine and 2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine were carried out, to elucidate whether benzylamino-1,3,5-triazines use the same binding niche as atrazine. [14C]-Atrazine and [7-14C]-2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine bound to wild-type thylakoids were replaced by non-labeled 2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine and non-labeled atrazine, respectively. The above two replacements showed mutual competition. To clarify further whether benzylamino-1,3,5-triazines bind at the D1-protein to amino acid residue(s) different from atrazine or not, experiments to replace [7-14C]-2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazines bound to atrazine-resistant Chenopodium thylakoids by non-labeled atrazine, 2-(4-bromobenzylamino)-4-methyl-6-trifluoromethyl-1,3,5-triazine, DCMU and DNOC were carried out. Although the bound [7-14C]-2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine was difficult to be replaced even with high concentrations of atrazine, [14C]-labeled 1,3,5-triazine was competitively replaced by non-labeled 2-(4-bromobenzylamino)-4-methyl-6-trifluoromethyl-1,3,5-triazine, DCMU or DNOC. Thus, 2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine herbicides are considered to bind to the same niche at the D1 protein as atrazine, but use amino acid residue(s) different from those involved with atrazine binding.

Chlorophyll fluorescence measurements have a wide range of applications from basic understanding of photosynthesis functioning to plant environmental stress responses and direct assessments of plant health. The measured signal is the fluorescence intensity (expressed in relative units) and the most meaningful data are derived from the time dependent increase in fluorescence intensity achieved upon application of continuous bright light to a previously dark adapted sample. The fluorescence response changes over time and is termed the Kautsky curve or chlorophyll fluorescence transient. Recently, Strasser and Strasser (1995) formulated a group of fluorescence parameters, called the JIP-test, that quantify the stepwise flow of energy through Photosystem II, using input data from the fluorescence transient. The purpose of this study was to establish relationships between the biochemical reactions occurring in PS II and specific JIP-test parameters. This was approached using isolated systems that facilitated the addition of modifying agents, a PS II electron transport inhibitor, an electron acceptor and an uncoupler, whose effects on PS II activity are well documented in the literature. The alteration to PS II activity caused by each of these compounds could then be monitored through the JIP-test parameters and compared and contrasted with the literature. The known alteration in PS II activity of Chenopodium album atrazine resistant and sensitive biotypes was also used to gauge the effectiveness and sensitivity of the JIP-test. The information gained from the in vitro study was successfully applied to an in situ study. This is the first in a series of four papers. It shows that the trapping parameters of the JIP-test were most affected by illumination and that the reduction in trapping had a run-on effect to inhibit electron transport. When irradiance exposure proceeded to photoinhibition, the electron transport probability parameter was greatly reduced and dissipation significantly increased. These results illustrate the advantage of monitoring a number of fluorescence parameters over the use of just one, which is often the case when the FV/FM ratio is used.

The effects of Photosystem II inhibiting herbicides, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron), atrazine and two novel 2-benzylamino-1,3,5-triazine compounds, on photosynthetic oxygen evolution and chlorophyll a fluorescence induction were measured in thylakoids isolated from Chenopodium album (wild type and atrazine-resistant plants) and cyanobacterial intact cells. The resistant plants have a mutation of serine for glycine at position 264 of the D1 protein. Diuron and two members of a novel class of 2-benzylamino-1,3,5-triazine compounds were almost as active in wild-type as in atrazine-resistant thylakoids, indicating that the benzylamino substitution in the novel triazines may be important for the lack of resistance in these atrazine-resistant plants. The inhibition by the herbicides of oxygen evolution in the cyanobacteria was somewhat lower than in the thylakoids of Chenopodium album wild type, probably caused by a slower uptake in the intact cells. The so-called OJIP fluorescence induction curve was measured during a one second light pulse in the absence and in the presence of high concentrations of the four herbicides. In the presence of a herbicide we observed an increase of the initial fluorescence at the origin (Fo′), a higher J level, and a decreased steady state at its P level (Fp). The increase to Fo′ and the decreased leveling Fp are discussed. After dark adaptation about 25% of the reaction centers are in the S0 state of the oxygen evolving complex with an electron on the secondary electron accepting quinone, QB. The addition of a herbicide causes a transfer of the electron on QB to the primary quinone acceptor, QA, and displacement of QB by the herbicide; the reduced QA leads to a higher Fo′. The decrease of Fp in the presence of the herbicides is suggested to be caused by inhibition of the photo-electrochemical stimulation of the fluorescence yield.

Besides being the substrate for the carboxylation reaction of photosynthesis, CO2 (bicarbonate) is required for the activity of Photosystem II (water plastoquinone oxido-reductase). It plays a role on the electron donor side as well as the electron acceptor side. In this contribution, attention will mostly be focused on the history of research into the effects of bicarbonate on electron flow reactions on the acceptor side. Donor side reactions are discussed in this issue by Alan Stemler.

The effects of nine novel 2-benzylamino-1,3,5-triazines on photosynthetic reactions were measured in thylakoids isolated from wild-type and atrazine-resistant plants of Chenopodium album. The resistant plants have a mutation of serine for glycine at position 264 of the D1 protein. The measurement of oxygen evolution and chlorophyll a fluorescence induction indicated a 2–4-fold stronger inhibition by the 6-trifluoromethyl analogues of Photosystem II-dependent electron flow than atrazine. Analogues having a 6-methyl-, 6-monofluoromethyl or 6-difluoromethyl substitution were weak inhibitors, indicating that the 6-trifluoro group is very important for strong inhibition. All the nine novel 2-benzylamino-1,3,5-triazines were almost as active in wild-type as in atrazine-resistant thylakoids, indicating that the benzylamino substitution may be important for the lack of resistance in the atrazine-resistant plants.

Effects of photoinhibition on photosynthesis in pea (Pisum sativum L.) leaves were investigated by studying the relationship between the severity of a photoinhibitory treatment (measured as Fv/Fm) and several photoacoustic and chlorophyll a fluorescence parameters. Because of the observed linear relationship between the decline of Fv/Fm and the potential oxygen evolution rate determined by the photoacoustic method, the parameter Fv/Fm was used as an indicator for the severity of photoinhibition. Our analysis revealed that part of the Photosystem II (PS II) reaction centers is inactive in oxygen evolution and is also less sensitive to photoinhibition. Correcting the parameter qP (fraction of open PS II reaction centers) for inactive PS II centers unveiled a strong increase of qP in severely inhibited pea leaves, indicating that the inactivated active centers do no longer contribute to qP and that photoinhibition has an all or none effect on PS II centers. Analysis of qE (energy quenching) demonstrated its initial increase possibly associated with dephosphorylation of LHC II. Analysis of qI (photoinhibition dependent quenching) showed that the half-time of recovery of qI increases steeply below an Fv/Fm of 0.65. This increase of the relaxation half-time corresponds with a decrease of the electron transport rate J and tentatively indicates that the supply of ATP, needed for the recovery, starts to decrease. The data indicate the necessity of correcting for inactive centers in order to make valuable conclusions about effects of photoinhibition on photosynthetic parameters.

The photosynthetic apparatus can be damaged by light energy in the process of photoinhibition. The target of this photoinhibition is mainly photosystem II (PSII). The mechanism leading to photoinhibitory damage is not yet known. We have characterized photoinhibition by measuring the photoinactivation of electron transport rates using the electron acceptors silicomolybdate and ferricyanide at different irradiance levels and different pH values. The effects of light on the donor side of PSII were measured with silicomolybdate, the effects on the acceptor side were measured with ferricyanide. We observed that photoinactivation of both donor and acceptor side of PSII are light dose-dependent, donor and acceptor side inactivation being independent processes. The donor side of PSII is less sensitive to photoinhibition than the acceptor side. The difference in pH dependence of donor and acceptor side photoinactivation leads us to propose that light-induced release of bicarbonate from PSII is a primary event leading to photoinhibition. In addition, we report that a photoinhibitory treatment increases the proton permeability of thylakoid membranes. This increase seems to be related to the presence of inactivated PSII reaction centers. It is suggested that radicals formed by inactivated PSII reaction centers causing lipid peroxidation are responsible.

The adaptive ability of thylakoid membranes in response to a stress given in the form of an in situ prolonged partial inhibition of PSII electron transport activity was analyzed. The membranes showed alterations in the structure-function relationship, the most prominent being, an increase in the antenna size of PSII. Analysis of chlorophyll fluorescence decay following a single turnover saturating flash and electrochromic shift measurements suggest that an increase in the unit size of PSII was accompanied by a small increase in the number of PSII units in treated plants; the ratio of active PSII to inactive PSII, however, remained unaffected. The partern of excitation energy distribution between the photosystems was altered in the thylakoids of treated plants such that energy transfer from PSII to PSI (spillover) increased. A physiological significance of such alterations is suggested according to which the partial inhibition of PSII electron transport is compensated for by an increase in the light harvesting capacity of photosystem II due to a larger antenna size and also by an increase in the number of photosystem units.

In oxygenic photosynthesis, carbon dioxide is fixed by ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and further reduced to carbohydrates. However, CO2, in the form of carbonate or bicarbonate, is also directly involved in the “light reactions” through structural and regulatory roles within Photosystem II (PS II). A notable feature is antagonistic interactions between bicarbonate (carbonate) and monovalent anions such as formate within PS II. Incubation of PS II-containing samples with formate results in the inhibition of electron flow activity, which can be restored only by the addition of bicarbonate. This “bicarbonate effect” influences molecular processes associated with both the electron acceptor and electron donor sides of PS II. The bicarbonate interaction on the acceptor side is located in the region of the primary and secondary quinones and contributes to the protonation states associated with quinol formation. At physiological pH, bicarbonate (carbonate) is a ligand to the non-heme iron and forms hydrogen bonds to several amino acids of the D1 and D2 proteins. Bicarbonate may stabilize, through conformational means, the reaction center proteins by protonation of certain amino acids near the secondary quinone electron acceptor. A possible functional role in vivo is that it controls PS II electron flow in order to ameliorate the impact of stress conditions leading to, for instance, photoinhibition or thermoinactivation. The role of bicarbonate on the donor of PS II has been the subject of renewed interest and bicarbonate has been suggested to play a role in the assembly of the Mn4Ca cluster during photoactivation. Additionally, a role as a catalytic base or proton transporter on the donor side of PS II has been proposed. However, while clear evidence for bicarbonate’s role on the acceptor side has been established, experiments designed to elucidate the putative role of bicarbonate on the donor side of PS II have not yet provided convincing evidence.

Plants of wild-type and triazine-resistant Canola (Brassica napus L.) were exposed to very high light intensities and after 1 day placed on a laboratory table at low light to recover, to study the kinetics of variable fluorescence after light, and after dark-adaptation. This cycle was repeated several times. The fast OJIP fluorescence rise curve was measured immediately after light exposure and after recovery during 1 day in laboratory room light. A fluorescence induction algorithm has been used for resolution and analysis of these curves. This algorithm includes photochemical and photo-electrochemical quenching release components and a photo-electrical dependent IP-component. The analysis revealed a substantial suppression of the photo-electrochemical component (even complete in the resistant biotype), a partial suppression of the photochemical component and a decrease in the fluorescence parameter F (o) after high light. These effects were recovered after 1 day in the indoor light.

The effects of a photoinhibition treatment (PIT) on electron transport and photophosphorylation reactions were measured in chloroplasts isolated from triazine-resistant and susceptible Chenopodium album plants grown under high and low irradiance. Electron transport dependent on photosystem I (PSI) alone was much less affected by PIT than that dependent on both photosystem II (PSII) and PSI. There was a smaller difference in susceptibility to PIT between the photophosphorylation activitity dependent on PSI alone and that dependent on both PSII and PSI. Because in all cases photophosphorylation activity decreased faster upon PIT than the rate of electron transport, we conclude that photoinhibition causes a gradual uncoupling of electron transport with phosphorylation. Since the extent of the light-induced proton gradient across the thylakoid membrane decreased upon PIT, it is suggested that photoinhibiton causes a proton leakiness of the membrane. We have found no significant differences to PIT of the various reactions measured in chloroplasts isolated from triazine-resistant and susceptible plants. We have also not observed any significant differences to PIT of the photophosphorylation reactions in chloroplasts of plants grown under low irradiance, compared with those grown under high irradiance. However, the electron transport reactions in chloroplasts from plants grown under low irradiance appeared to be somewhat less sensitive to PIT than those grown under high irradiance.

Plants resistant to triazine-type herbicides are known to be altered in their photosystem II reaction center. Serine at site 264 in D1 protein is replaced by glycine. The measurements of chlorophyll a fluorescence excitations with a variable number of saturating flashes in Chenopodium album plants show characteristic differences between the resistant and the wild-type plants. These differences appear in response to the first flash as well as in the rise pattern of subsequent flashes of a 12.5 Hz flash train. The differences indicate a higher concentration of Q(B)-nonreducing reaction centers in the resistant biotype, and confirm earlier results on a slower rate of electron transport between the primary and secondary electron acceptors.

The effects of ultraviolet-B (UV-B) radiation on photosystem II (PS II) were studied in leaves of Chenopodium album. After the treatment with UV-B the damage was estimated using chlorophyll a fluorescence techniques. Measurements of modulated fluorescence using a pulse amplitude modulated fluorometer revealed that the efficiency of photosystem II decreased both with increasing time of UV-B radiation and with increasing intensity of the UV-B. Fluorescence induction rise curves were analyzed using a mechanistic model of energy trapping. It appears that the damage by UV-B radiation occurs first at the acceptor side of photosystem II, and only later at the donor side.

The effects of Photosystem II inhibiting herbicides, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron), atrazine and two novel 2-benzylamino-1,3,5-triazine compounds, on photosynthetic oxygen evolution and chlorophyll a fluorescence induction were measured in thylakoids isolated from Chenopodium album (wild type and atrazine-resistant plants) and cyanobacterial intact cells. The resistant plants have a mutation of serine for glycine at position 264 of the D1 protein. Diuron and two members of a novel class of 2-benzylamino-1,3,5-triazine compounds were almost as active in wild-type as in atrazine-resistant thylakoids, indicating that the benzylamino substitution in the novel triazines may be important for the lack of resistance in these atrazine-resistant plants. The inhibition by the herbicides of oxygen evolution in the cyanobacteria was somewhat lower than in the thylakoids of Chenopodium album wild type, probably caused by a slower uptake in the intact cells. The so-called OJIP fluorescence induction curve was measured during a one second light pulse in the absence and in the presence of high concentrations of the four herbicides. In the presence of a herbicide we observed an increase of the initial fluorescence at the origin (Fo'), a higher J level, and a decreased steady state at its P level (Fp). The increase to Fo' and the decreased leveling Fp are discussed. After dark adaptation about 25% of the reaction centers are in the S(0) state of the oxygen evolving complex with an electron on the secondary electron accepting quinone, Q(B). The addition of a herbicide causes a transfer of the electron on Q(B) to the primary quinone acceptor, Q(A), and displacement of Q(B) by the herbicide; the reduced Q(A) leads to a higher Fo'. The decrease of Fp in the presence of the herbicides is suggested to be caused by inhibition of the photo-electrochemical stimulation of the fluorescence yield.

Besides being the substrate for the carboxylation reaction of photosynthesis, CO(2) (bicarbonate) is required for the activity of Photosystem II (water plastoquinone oxido-reductase). It plays a role on the electron donor side as well as the electron acceptor side. In this contribution, attention will mostly be focused on the history of research into the effects of bicarbonate on electron flow reactions on the acceptor side. Donor side reactions are discussed in this issue by Alan Stemler.

Chlorophyll fluorescence measurements have a wide range of applications from basic understanding of photosynthesis functioning to plant environmental stress responses and direct assessments of plant health. The measured signal is the fluorescence intensity (expressed in relative units) and the most meaningful data are derived from the time dependent increase in fluorescence intensity achieved upon application of continuous bright light to a previously dark adapted sample. The fluorescence response changes over time and is termed the Kautsky curve or chlorophyll fluorescence transient. Recently, Strasser and Strasser (1995) formulated a group of fluorescence parameters, called the JIP-test, that quantify the stepwise flow of energy through Photosystem II, using input data from the fluorescence transient. The purpose of this study was to establish relationships between the biochemical reactions occurring in PS II and specific JIP-test parameters. This was approached using isolated systems that facilitated the addition of modifying agents, a PS II electron transport inhibitor, an electron acceptor and an uncoupler, whose effects on PS II activity are well documented in the literature. The alteration to PS II activity caused by each of these compounds could then be monitored through the JIP-test parameters and compared and contrasted with the literature. The known alteration in PS II activity of Chenopodium album atrazine resistant and sensitive biotypes was also used to gauge the effectiveness and sensitivity of the JIP-test. The information gained from the in vitro study was successfully applied to an in situ study. This is the first in a series of four papers. It shows that the trapping parameters of the JIP-test were most affected by illumination and that the reduction in trapping had a run-on effect to inhibit electron transport. When irradiance exposure proceeded to photoinhibition, the electron transport probability parameter was greatly reduced and dissipation significantly increased. These results illustrate the advantage of monitoring a number of fluorescence parameters over the use of just one, which is often the case when the F(V)/F(M) ratio is used.

A series of replacement experiments of [(14)C]-triazines, [(14)C]-atrazine and [7-(14)C]-2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine, bound to thylakoids isolated from wild-type and atrazine-resistant Chenopodium album (lambsquarters) were conducted. Replacement experiments of [(14)C]-triazines bound to wild-type Chenopodium thylakoids with non-labeled atrazine and 2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine were carried out, to elucidate whether benzylamino-1,3,5-triazines use the same binding niche as atrazine. [(14)C]-Atrazine and [7-(14)C]-2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine bound to wild-type thylakoids were replaced by non-labeled 2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine and non-labeled atrazine, respectively. The above two replacements showed mutual competition. To clarify further whether benzylamino-1,3,5-triazines bind at the D1-protein to amino acid residue(s) different from atrazine or not, experiments to replace [7-(14)C]-2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazines bound to atrazine-resistant Chenopodium thylakoids by non-labeled atrazine, 2-(4-bromobenzylamino)-4-methyl-6-trifluoromethyl-1,3,5-triazine, DCMU and DNOC were carried out. Although the bound [7-(14)C]-2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine was difficult to be replaced even with high concentrations of atrazine, [(14)C]-labeled 1,3,5-triazine was competitively replaced by non-labeled 2-(4-bromobenzylamino)-4-methyl-6-trifluoromethyl-1,3,5-triazine, DCMU or DNOC. Thus, 2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine herbicides are considered to bind to the same niche at the D1 protein as atrazine, but use amino acid residue(s) different from those involved with atrazine binding.

This study relates to the modification of 2-benzylamino-4-methyl-6-trifluoromethyl-1,3,5-triazine. New 1,3,5-triazine compounds with an electron-withdrawing carboxyl group, e.g. ester group, substituted for the trifluoromethyl group, were synthesized and assayed for activity to inhibit photosynthetic electron transport (PET) in thylakoids of spinach as well as both atrazine-resistant and wild-type Chenopodium album. Among the compounds with an alkylamino group, 2-ethoxycarbonyl-4-isopropylamino-6-methyl-1,3,5-triazine was the most potent PET-inhibitor, exhibiting a pI50 of 6.11. The inhibitory activity was generally more potent with 2-alkoxycarbonyl-4-(branched alkyl)amino-6-methyl-1,3,5-triazines than amino-analogues with straight chain alkyl groups or unsaturated alkyl groups. © Presticide Science Society of Japan

Chlorophyll fluorescence induction curves of isolated thylakoids were measured in the absence and in the presence of various concentrations of photosystem II-inhibiting herbicides. A mathematical program was applied to simulate the curves. Based on these simulated curves a new method is developed to determine the concentration effect (pI(50)) of the herbicides. The results of the new method correspond well with reported values in the literature. The method is very convenient and time-saving. (C) 2004 Elsevier Inc. All rights reserved.

Depletion of bicarbonate (carbon dioxide) from oxygenic cells or organelles not only causes cessation of carbon dioxide fixation, but also a strong decrease in the activity of photosystem II; the photosystem II activity can be restored by readdition of bicarbonate. Effects of bicarbonate exist on both the acceptor as well as on the donor side of photosystem II. The influence on the acceptor side is located between the primary and secondary quinone electron acceptor of photosystem II, and can be demonstrated in intact cells or leaves as well as in isolated thylakoids and reaction center preparations. At physiological pH, bicarbonate ions are suggested to form hydrogen bonds to several amino acids on both D1 and D2 proteins, the reaction center subunits of photosystem II, as well as to form ligands to the non-heme iron between the D1 and D2 proteins. Bicarbonate, at physiological pH, has an important role in the water-plastoquinone oxido-reductase: on the one hand it may stabilize, by conformational means, the reaction center protein of photosystem II that allows efficient electron flow and protonation of certain amino acids near the secondary quinone electron acceptor of photosystem II; and, on the other hand, it akppears to play a significant role in the assembly or functioning of the manganese complex at the donor side. Functional roles of bicarbonate in vivo, including protection against photoinhibition, are also discussed.

Empirical parameters derived from the Chlorophyll-a fluorescence transient have long been used to investigate the photosynthetic performance of plants. This transient, when plotted on a logarithmic time scale, yields a polyphasic signal, termed the OJIP fluorescence transient [1] — (see Fig. 1). However, a full interpretation of the photosynthetic reactions underlying this transient based on experimental data or theoretical analyses, is unavailable. In order to fully utilise this sensitive, non-invasive probe into in vivo photosynthesis, the inherent complexity of the fluorescence transient must be unravelled.

One of the first reactions of a plant upon exposure to abiotic stress is a lowered activity of photosystem II (PSII). Therefore it has become very popular to estimate the effects of abiotic stress on plants with the aid of fluorescence methods: the measurement of chlorophyll a fluorescence emitted by PSII as Fv/Fm. However, the photoprotective mechanisms leading to fluorescence emission by PSII are still insufficiently understood. Even less is known about the adaptation of plants to abiotic stress.

The two-electron gate of Photosystem II (PSII) is known to function by transferring electrons from the reduced one electron acceptor Q A − (a bound plastosemiquinone) to the oxidized two-electron acceptor Q B (a bound plastoquinone), and then again from Q A − to the singly reduced Q B − , producing plastoquinol Q B H 2 . In this article, we have three chloroacetates (monochloroacetate, MCA; dichloroacetate, DCA; and trichloroacetate, TCA), having different geometry and hydrophobicity, to probe the binding environment of the two-electron gate in spinach thylakoids. We first established that these chloroacetates up to 100 mM act specifically in the Q A Q B region by monitoring partial reactions of PSII as well as PSI, measuring thermoluminescence, specific for recombination reactions between the donor and acceptor sides of PSII, and studying chlorophyll (Chl) a fluorescence decay in the micro to millisecond region, specific for electron flow from Q A − to Q B . Further, the site of action was located on the D1–D2 protein through observations on the differential sensitivity of chloroacetates on specific D1–D2 mutants of the cyanobacterium Synechocystis sp. PCC 6803. Detailed measurements were then done to characterized the effect of chloroacetates on Q A Q B reactions. Data on the [Q A − ] decay kinetics led to the following observations: (1) chloroacetates (and acetate) not only increase the time constant of electron flow from Q A − to Q B (Q B − ), but increase the equilibrium [Q A − ] both after flash 1 and 2, and the degree of these effects (lowest to highest) is correlated with the geometry (increased number of chlorine moiety) and increase hydrophobicity of these inhibitors: the hierarchy is: acetate A − ] equilibrium with DCA and TCA. At pH 7.5 however, flash 1 effects were larger than flash 2 effects with all chloroacetates. (3) Bicarbonate reverses the inhibitory effect on Q A − to Q A (Q B − ) reactions also in a differentimanner; the hierarchy for the most reversible (or least irrversible) is: acetate ⪆ MCA > DCA ⪢ TCA (4) The pH dependence of the inhibitory effects on Q A − to Q B (Q B − )are: te MCA and DCA effects are larger at pH6 than at pH 7.5, but the TCA effects are higher at pH 7.5 than at pH 6. The above results, taken together with those in the literature, are in agreement with a picture of the Q A FeQ B niche in the D1–D2 protein of PSII where quinones, herbicides, chloroacetates, formate as well as bicarbonate bind, but differently with different overlapping sites. Chloroacetates show effects on the two-electron gate that place them ‘in between’ the herbicides and the bicarbonate-reversible formate.

Molecular mechanisms of photoinhibition are clarified on the basis of experiments with leaves and isolated chloroplasts of peas and triazine-resistant and susceptible Chenopodium plants.

The photosystem II reaction center can be considered as a water-plastoquinone oxido-reductase. Using four photons it transfers four electrons from two molecules of water to plastoquinone producing molecular oxygen and two molecules of doubly reduced plastoquinone. Our understanding of the structure and function of this complex has greatly increased during the recent years. The basis of the reaction center of photosystem II is formed by the DI and D2 proteins, both having a molecular mass of about 32 kDa. The DI protein contains not only the binding site for the physiological electron carrier QB, but also the binding sites for several classes of herbicides and for bicarbonate and formate.

Photosystem II herbicides bind to the D1 protein of the reaction centre of photosystem II. Resistance to these herbicides in plants is confined almost exclusively to the triazine group and involves an altered D1 protein: at site 264 the serine of the wild type is replaced by a glycine. The electron flow rate between the primary electron acceptor of photosystem II and the plastoquinone pool is about three times slower in triazine-resistant plants, but the overall electron transport rate is not different from that in sensitive plants. When plants are grown at a low light intensity, there is no difference in photosynthetic capacity and 1n fitness. However, when grown at a high light intensity, resistant plants have a lower photosynthetic capacity and are less fit. This difference is ascribed to a higher sensitivity of resistant plants to photoinhibition. Measurements of the reversible binding and release kinetics revealed that the binding kinetics are not much different in sensitive and resistant plants. Triazine resistance appears to originate from a significant increase of the release kinetics.

In isolated broken chloroplasts photosynthetic electron transport requires the presence of CO2 and/or bicarbonate. This bicarbonate effect on electron flow was measured in a medium containing 100 mM sodium formate. In this medium a dark incubation time with bicarbonate is required for the reactivation of the Hill reaction. We have measured the kinetics of the reactivation of electron flow by varying the dark incubation of CO2-depIeted pea (Pisum sativum L., cv. Rondo) chloroplasts with bicarbonate. The half-time of this reactivation appears to be 25 s when 2 mM bicarbonate is added. The dinitrophenol herbicide, i-dinoseb, is shown to be a competitive inhibitor of the bicarbonate dependent Hill reaction with an inhibitor constant (Ki) of 31 nM. In the presence of 100 nM i-dinoseb or 100 nM DCMU the half-time of the reactivation by 2 mM bicarbonate appears to increase to about 58 s. We provide an explanation for these phenomena by analyzing the bicarbonate-thylakoid interaction on the basis of a simple reaction scheme. The binding of bicarbonate to the thylakoids appears to be a second order reaction with pseudo-first order kinetics. According to our analysis, any inhibitor, which is competitive with respect to the bicarbonate stimulation of the Hill reaction, should increase the half-time of the reactivation of the Hill reaction.

The heterogeneity of photosystem II with respect to α and β centers was investigated in triazine-resistant and susceptible biotypes of Chenopodium album . In both biotypes the light harvesting antenna sizes of photosystem II α centers was larger than those of β centers. In the resistant biotype the antenna size of the α centers was smaller than those in the susceptible one. There was not much difference in the antenna sizes of the β centers. The proportion of β centers was larger in the resistant biotype compared with the sensitive one.

Bicarbonate (or carbon dioxide) is required for electron transport in isolated broken pea chloroplasts. The site of action of the bicarbonate ion is between the primary electron acceptor of Photosystem 2, Q, and the plastoquinone pool. After trypsin treatment the Hill reaction with ferricyanide does not require bicarbonate. Photosystem 2 inhibiting herbicides act also at this site. Therefore, a possible interaction of bicarbonate and these herbicides in their effect on photosynthetic electron transport was studied. The reciprocal of the Hill reaction rate in CO2-depleted chloroplasts was plotted against the reciprocal of added bicarbonate concentration in the absence and in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), 2-methoxy-4,6-bis (ethylamino)-1,3,5-triazine (simeton) or 4,6-dinitro-o-cresol (DNOC). From these Lineweaver-Burk plots we concluded that DCMU and simeton inhibit both bicarbonate binding and Vmax. There is a purely competitive inhibition of bicarbonate binding by DNOC. We suggest that DNOC may exert its inhibition of electron transport by removing bicarbonate from its binding site. In isolated thylakoid membranes of Synechococcus leopoliensis we did not find a bicarbonate effect nor inhibition by DNOC after Q, indicating that in the thylakoids of this blue-green alga the binding site for bicarbonate and DNOC between Q and plastoquinone is absent.

The photosynthetic oxygen evolution is schematically described by the model of Kok et al. [1]. An excited PS II reaction center is able to extract electrons from the oxygen evolving complex (OEC) in a 4-step sequence. The evolution of 1 molecule of O2 requires cooperation of 4 oxidizing equivalents. The OEC contains 4 Mn atoms, which can successively be oxidized and thus accommodate positive charges.

In CO2-depleted chloroplasts electron transport between the Photosystem II electron acceptor Q and plastoquinone is largely suppressed. In the presence of a high concentration of sodium formate (greater than 10 mM), which probably binds to the bicarbonate site, addition of bicarbonate restores the ferricyanide Hill reaction only after incubation in the dark. With lower formate concentrations bicarbonate is able to restore electron transport in the light. The Hill reaction rate in CO2-depleted chloroplasts after bicarbonate addition, divided by the rate in CO2-depleted chloroplasts before bicarbonate addition, shows a sharp optimum at pH 6.5. Furthermore, the rate-limiting step in bicarbonate action is probably diffusion. The results are explained in terms of a hypothetical model: the bicarbonate-binding site is located at the outer side of the thylakoid membrane, but not directly accessible from the ‘bulk’. To reach the site from the bulk, the molecule has to pass a channel with negatively charged groups on its side walls. In the light these groups are more negatively charged than in the dark. Therefore, the formate ion cannot exchange for bicarbonate in the light, and a dark period is necessary to enable exchange of formate for bicarbonate.

1. 1. CO2-depletion of thylakoid membranes results in a decrease of binding affinity of the Photosystem II (PS II) inhibitor atrazine. The inhibitory efficiency of atrazine, expressed as I50-concentration (50% inhibition) of 2,6-dichlorophenolindophenol reduction, is the same in CO2-depleted as well as in control thylakoids. This shows that CO2-depletion results in a complete inactivation of a part of the total number of electron transport chains. 2. 2. A major site of action of CO2, which had previously been located between the two electron acceptor quinone molecule B (or R) and plastoquinone, appears to be in the same membrane protein which binds the Photosystem II inhibitor atrazine as suggested by the following observations: (a) CO2-depletion results in a shift of the binding constant (kb) of [14C]atrazine to thylakoid mebranes indicating a decreased affinity of atrazine to the membrane; (b) trypsin treatment, which is known to modify the Photosystem II complex at the level of B, strongly diminishes CO2 stimulation of electron transport reactions in CO2-depleted membranes; and (c) thylakoids from atrazine-resistant plants, which contain a Photosystem II complex modified at the inhibitor binding site, show an altered CO2-stimulation of electron flow. 3. 3. CO2-depletion does not produce structural changes in enzyme complexes involved in Photosystem II function of thylakoid membranes, as shown by freeze-fracture studies using electron microscopy.

Many commercially available herbicides block the photosynthetic electron transport of plants, by preventing the oxidation of the primary acceptor QA of Photosystem II by the secondary acceptor QB. Compounds from different chemical classes, as e.g. DCMU, atrazine and dinoseb, all bind to the same protein. This QB-protein, with MW = 32000 dalton, is located on the acceptor side of PS II. It is part of a binding environment for two different plastoquinone molecules, the tightly bound QA and the easier exchanged QB (1). This QB can accept 2 electrons, and then becomes proton-ated to QBH2・ Both the oxidized and the fully reduced form are rapidly (50 s−1) exchanging with free plastoquinone molecules from the PQ-pool, but the semi-quinone form is not (2,3).