Interaction of Herbicides and Quinone with the Q(B)-Protein of the Diuron-Resistant Chlamydomonas reinhardtii Mutant Dr2

We have used the diuron-resistant Dr2 mutant of Chlamydomonas reinhardtii which is altered in the 32 kilodalton Q_B-protein at amino acid 219 (valine to isoleucine), to investigate the interactions of herbicides and plastoquinone with the 32 kilodalton Q_B-protein. The data contained in this report demonstrate that the effects of this mutation are different from those of the more completely characterized mutant which confers extreme resistance to triazines in higher plants. The mutation in C. reinhardtii Dr2 confers only slight resistance to a number of inhibitors of photosynthetic electron transport. Extreme triazine resistance results from an increase in the binding constant of the herbicide with the 32 kilodalton Q_B-protein, in contrast the diuron binding constant for chloroplasts isolated from wild-type (sensitive) Chlamydomonas and the resistant Dr2 are indistinguishable. We conclude that the altered structure in the 32 kilodalton Q_B-protein of Dr2 does not directly affect the diuron binding site. This mutation appears to alter the steric properties of the binding protein in such a way that diuron and plastoquinone do not directly compete for binding. This steric perturbation confers mild resistance to other herbicidal inhibitors of photosynthesis and alters the kinetics of Q_A to Q_B electron transfer.     

The mechanism of herbicide resistance in tobacco cells with a new mutation in the q(b) 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.     

Kinetics of photosystem II electron transport: a mathematical analysis based on chlorophyll fluorescence induction

The OJDIP rise in chlorophyll fluorescence during induction at different light intensities was mathematically modeled using 24 master equations describing electron transport through photosystem II (PSII) plus ordinary differential equations for electron budgets in plastoquinone, cytochrome f, plastocyanin, photosystem I, and ferredoxin. A novel feature of the model is consideration of electron in- and outflow budgets resulting in changes in redox states of Tyrosine Z, P680, and Q_A as sole bases for changes in fluorescence yield during the transient. Ad hoc contributions by transmembrane electric fields, protein conformational changes, or other putative quenching species were unnecessary to account for primary features of the phenomenon, except a peculiar slowdown of intra-PSII electron transport during induction at low light intensities. The lower than F _m post-flash fluorescence yield F _f was related to oxidized tyrosine Z. The transient J peak was associated with equal rates of electron arrival to and departure from Q_A and requires that electron transfer from Q_A ^? to Q_B be slower than that from Q_A ^? to Q_B ^?. Strong quenching by oxidized P680 caused the dip D. Reduced plastoquinone, a competitive product inhibitor of PSII, blocked electron transport proportionally with its concentration. Electron transport rate indicated by fluorescence quenching was faster than the rate indicated by O₂ evolution, because oxidized donor side carriers quench fluorescence but do not transport electrons. The thermal phase of the fluorescence rise beyond the J phase was caused by a progressive increase in the fraction of PSII with reduced Q_A and reduced donor side.     

Paternal inheritance of mitochondria in rapeseed (Brassica napus)

Summary Transfer of a mitochondrially associated plasmid following sexual crosses in Brassica napus rapeseed suggested that paternal mitochondria were being transferred to the cytoplasm of the egg. To examine this possibility further, plants carrying the chloroplast (cp) marker of triazine resistance, but which had lost the plasmid associated with the mitochondria of this cytoplasm, were crossed as females to males carrying the polima cytoplasm. The males carried a nuclear fertility restorer gene on an extra chromosome to overcome the male sterility marker conferred by the mitochondria of this cytoplasm. Approximately 10% of the F₁ progeny displayed the male sterility and flower morphology of the male parent. Mitochondrial (mt) DNA from the progeny showed the combined restriction patterns of both parents, but this rut heterogeneity did not continue into subsequent generations. All progeny retained the cp DNA restriction patterns of the maternal plant as well as resistance to the herbicide atrazine. To date, sexually mediated cybrid plants have shown no morphological abnormalities and have maintained their unique combination of cp and mt traits through several sexual generations.     

PSII Complexes with Destabilized Primary Quinone Acceptor of Electrons in Dark-Adapted Chlorella

Incubation of the alga Chlorella pyrenoidosa Chick in darkness (at 37°C) for 24 h did not change the initial (F ₀) and maximum (F _m) yield of chlorophyll fluorescence in diuron-treated cells. In dark-incubated alga, the contribution of the slow (rise time 10–15 min) phases to the kinetics of F _m rise and, correspondingly, to variable fluorescence F _v (where F _v = F _m – F ₀) increased twofold. In addition, F _m was attained at higher concentrations of diuron, which inhibits electron transfer between the primary (Q _A) and secondary (Q _B) quinone acceptors of electron in the PSII. Inhibition of photosynthetic electron transfer with o-phenanthroline, which, at high concentrations, competitively replaces both Q _B and Q _A, decreased F _m yield due to selective suppression of the slow phase of fluorescence rise. It was assumed that the slow phase in the kinetics of F _m rise reflects the functioning of PSII complexes with destabilized Q _A. Such destabilization can result from the modification of the major PSII proteins (D1 and D2) in dark-adapted Chlorella cells.     

Fluorescence characteristics of photoautotrophic soybean cells

We report here the first measurements on chlorophyll (Chl) a fluorescence characteristics of photoautotrophic soybean cells (cell lines SB-P and SBI-P). The cell fluorescence is free from severe distortion problems encountered in higher plant leaves. Chl a fluorescence spectra at 77 K show, after correction for the spectral sensitivity of the photomultiplier and the emission monochromator, peaks at 688, 696 and 745 nm, representing antenna systems of photosystem II-CP43 and CP47, and photosystem I, respectively. Calculations, based on the complementary area over the Chl a fluorescence induction curve, indicated a ratio of 6 of the mobile plastoquinone (including Q_B) to the primary stable electron acceptor, the bound plastoquinone Q_A. A ratio of one between the secondary stable electron acceptor, bound plastoquinone Q_B, and its reduced form Q_B ^- was obtained by using a double flash technique. Owing to this ratio, the flash number dependence of the Chl a fluorescence showed a distinct period of four, implying a close relationship to the S state of the oxygen evolution mechanism. Analysis of the Q_A ^- reoxidation kinetics showed (1) the halftime of each of the major decay components ( 300 s fast and 30 ms slow) increases with the increase of diuron and atrazine concentrations; and (2) the amplitudes of the fast and the slow components change in a complementary fashion, the fast component disappearing at high concentrations of the inhibitors. This implies that the inhibitors used are able to totally displace Q_B. In intact soybean cells, the relative amplitude of the 30 ms to 300 s component is higher (40:60) than that in spinach chloroplasts (30:70), implying a larger contribution of the centers with unbound Q_B. SB-P and SBI-P soybean cells display a slightly different sensitivity of Q_A ^- decay to inhibitors.Abbreviations CA complementary area over fluorescence induction curve - Chl chlorophyll, diuron - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F _m maximum chlorophyll a fluorescence - F ₀ minimum chlorophyll a fluorescence - F _v = F _t-F₀ - where F _v = variable chlorophyll a fluorescence - and F_t = chlorophyll a fluorescence at time t - PS II photosystem II - Q _a primary (plastoquinone) electron acceptor of PS II - Q _b secondary (plastoquinone) electron acceptor of PS II - t₅₀ the time at which the concentration of reduced Q _a is 50% of that at its maximum value     

Sites of inhibition by disulfiram in thylakoid membranes

Disulfiram (tetraethylthiuram disulfide), a metal chelator, inhibits photosynthetic electron transport in broken chloroplasts. A major site of inhibition is detected on the electron-acceptor side of photosystem II between Q_A, the first plastoquinone electron-acceptor, and the second plastoquinone electron-acceptor, Q_B. This site of inhibition is shown by a severalfold increase in the half-time of Q⁻_A oxidation, as monitored by the decay of the variable chlorophyll a flourescence after an actinic flash. Another site of inhibition is detected in the functioning of the reaction center of photosystem II; disulfiram is observed to quench the room temperature variable chlorophyll a fluorescence, as well as the intensity of the 695 nm peak, relative to the 685 nm peak, in the chlorophyll a fluorescence spectrum at 77 K. Electron transport from H₂O to the photosystem II electron-acceptor silicomolybdate is also inhibited. Disulfiram does not inhibit electron flow before the site(s) of donation by exogenous electron donors to photosystem II, and no inhibition is detected in the partial reactions associated with photosystem I.     

Uptake of the Herbicide Diuron as Visualised by the Fluorescence Imaging Technique

A new fluorescence imaging system for monitoring the uptake of the PSII-herbicide diuron (OCMU) was tested in tobacco leaves. UV-laser-induced (Λ_exc = 355 nm) fluorescence images were collected for blue fluorescence F440 (Λ_em = 440 nm), green fluorescence F520 (Λ_em = 520 nm), red chlorophyll fluorescence F690 (Λ_em = 690 nm) and for far-red chlorophyll fluorescence F740 (Λ_em = 740 nm). Diuron-treated leaf parts exhibited a higher red and far-red chlorophyll fluorescence emission (F690 and F740) than untreated leaf halves, whereas the blue and green fluorescence, F440 and F520, remained unaffected. As a consequence, the fluorescence ratios blue/red (F440/F690) and blue/far-red (F440/F740) significantly decreased in diuron-treated leaf parts. The time course of diuron uptake into the leaf could be followed by fluorescence images taken 10 and 30 min after diuron application. The novel high resolution fluorescence imaging method supplies information on the herbicide uptake of each point of the leaf area. Its great advantage as compared to the point data fluorescence measurements applied so far is discussed.     

Loss of C-genome-specific markers during transgene introgression from Brassica napus to wild Brassica juncea

Transgene flow from engineered Brassica napus to wild weed relatives could potentially have an environmental effect. To evaluate the introgression of transgenic B. napus into wild Brassica juncea, the hybrid F₁ and backcross progenies derived from B. juncea (genome constitution AABB) and transgenic B. napus (AACC) crosses were investigated. C-genome-specific simple sequence repeat (SSR) markers corresponding to linkage groups N11–N19 in B. napus were screened and used to estimate the marker frequency in hybrid F₁ and backcross progenies. C-genome-specific markers could be stably detected in hybrid F₁ and backcross BC₁ plants, but were only rarely found in the BC₂–BC₅ generations. For example, a specific SSR marker for linkage group N12 segregated in BC₂ generation but were completely lost in BC₃–BC₅, while a specific SSR marker of linkage group N15 segregated in BC₁, BC₂ and BC₃ generations and was absent in more advanced backcrossed generations (BC₄ and BC₅). The results indicate that a certain gene regions in Brassica napus plants are transmitted at a relatively lower frequency to wild relatives, and more rapidly disappeared in subsequent backcross generations. We propose that a foreign gene or transgene that is integrated in the C-chromosome of Brassica napus could reduce the risk of introgression in nature.     

Energy fluxes and driving forces for photosynthesis in Lemna minor exposed to herbicides

Analysis of fast chlorophyll fluorescence rise OJIP was carried out to assess the impact of diuron, paraquat and flazasulfuron on energy fluxes and driving forces for photosynthesis in Lemna minor. Results showed that diuron and paraquat treatment produced major changes in electron transport in active reaction centres (RCs). However, diuron had a more pronounced effect on the yield of electron transport per trapped exciton (ψ₀) than on the yield of primary electron transport (φP₀)$({\phi }_{{\text{P}}_0})$ showing that dark reactions are more sensitive to diuron than light-dependent reactions. In contrast, paraquat treatment effects were not due to a target-specific action on those dark and light reactions. Paraquat also induced a marked surge in the total absorption of photosystem II (PSII) antenna chlorophyll per active RC displaying a large increase of the dissipation of excess energy through non-photochemical pathways (thermal dissipation processes). Flazasulfuron induced a slight decrease of both the total driving force for photosynthesis and the quantum yield of electron transport beyond Q_A⁻ combined to a small but significant increase of the non-photochemical energy dissipation per RC (DI₀/RC). We conclude that energy fluxes and driving force for photosynthesis generate useful information about the behaviour of aquatic plant photosystems helping to localize different target sites and to distinguish heterogeneities inside the PSII complexes. Regardless of the active molecule tested, the DF_ABS, φE₀${\phi }_{{\text{E}}_0}$, DI₀/RC and/or ET₀/RC parameters indicated a significant variation compared to control while φP₀${\phi }_{{\text{P}}_0}$ (F_V/F_M) showed no significant inhibition suggesting that those parameters are more sensitive for identifying a plant’s energy-use efficiency than the maximum quantum yield of primary PS II photochemistry alone.     

Transfer of auxinic herbicide resistance from Brassica kaber to Brassica juncea and Brassica rapa through embryo rescue

Auxinic herbicides are widely used in agriculture to selectively control broadleaf weeds. Prolonged use of auxinic herbicides has resulted in the evolution of resistance to these herbicides in some biotypes of Brassica kaber (wild mustard), a common weed in agricultural crops. In this study, auxinic herbicide resistance from B. kaber was transferred to Brassica juncea and Brassica rapa, two commercially important Brassica crops, by traditional breeding coupled with in vitro embryo rescue. A high frequency of embryo regeneration and hybrid plant establishment was achieved. Transfer of auxinic herbicide resistance from B. kaber to the hybrids was assessed by whole-plant screening of hybrids with dicamba, a widely used auxinic herbicide. Furthermore, the hybrids were tested for fertility (both pollen and pistil) and their ability to produce backcross progeny. The auxinic herbicide-resistant trait was introgressed into B. juncea by backcross breeding. DNA ploidy of the hybrids as well as of the backcross progeny was estimated by flow cytometry. Creation of auxinic herbicide-resistant Brassica crops by non-transgenic approaches should facilitate effective weed control, encourage less tillage, provide herbicide rotation options, minimize occurrence of herbicide resistance, and increase acceptance of these crops.     

Chimaeric CP47 mutants of the cyanobacterium Synechocystis sp. PCC 6803 carrying spinach sequences: Construction and function

Chimaeric mutants of the cyanobacterium Synechocystis sp. PCC 6803 have been generated carrying part or all of the spinach psbB gene, encoding CP47 (one of the chlorophyll-binding core antenna proteins in Photosystem II). The mutant in which the entire psbB gene had been replaced by the homologous gene from spinach was an obligate photoheterotroph and lacked Photosystem II complexes in its thylakoid membranes. However, this strain could be transformed with plasmids carrying selected regions of Synechocystis psbB to give rise to photoautotrophs with a chimaeric spinach/cyanobacterial CP47 protein. This process involved heterologous recombination in the cyanobacterium between psbB sequences from spinach and Synechocystis 6803; which was found to be reasonably effective in Synechocystis. Also other approaches were used that can produce a broad spectrum of chimaeric mutants in a single experiment. Functional characterization of the chimaeric photoautotrophic mutants indicated that if a decrease in the photoautotrophic growth rates was observed, this was correlated with a decrease in the number of Photosystem II reaction centers (on a chlorophyll basis) in the thylakoid membrane and with a decrease in oxygen evolution rates. Remaining Photosystem II reaction centers in these chimaeric mutants appeared to function rather normally, but thermoluminescence and chlorophyll a fluorescence measurements provided evidence for a destabilization of Q_B ^–. This illustrates the sensitivity of the functional properties of the PS II reaction center to mild perturbations in a neighboring protein.Abbreviations diuron 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F_v variable chlorophyll a fluorescence - HEPES N-(2-hydroxyethyl)piperazine-N-(2-ethanesulfonic acid) - (k)bp (kilo)base pairs - PS II Photosystem II - Q_A primary electron-accepting plastoquinone in Photosystem II - Q_B secondary electron-accepting plastoquinone in Photosystem II - SDS sodium dodecyl sulfate     

Mode of Inheritance of Amitraz Resistance in a Brazilian Strain of the Southern Cattle Tick, Boophilus microplus (Acari: Ixodidae)*

The southern cattle tick, Boophilus  microplus (Canestrini), has developed resistance to amitraz in several countries in recent years. A study was conducted at the USDA Cattle Fever Tick Research Laboratory in Texas to investigate the mode of inheritance of amitraz resistance with cross-mating experiments. The Muñoz strain, a laboratory reared acaricide-susceptible reference strain, was used as the susceptible parent and the Santa Luiza strain, originating in Brazil, was used as the resistant parent. A modified Food and Agriculture Organization Larval Packet Test was used to measure the levels of susceptibility of larvae of the parental strains, F₁, backcross, F₂, and F₃ generations. Results of reciprocal crossing experiments suggested that amitraz resistance was inherited as an incomplete recessive trait. There was a strong maternal effect on larval progeny’s susceptibility to amitraz in both the F₁ and the subsequent generations. The values of the degree of dominance were estimated at ?0.156 and ?0.500 for the F₁ larvae with resistant and susceptible female parents, respectively. Results of bioassays on larval progeny of the F₁ backcrossed with the resistant parent strain and that of the F₂ generations suggested that more than one gene was responsible for amitraz resistance in the Santa Luiza strain. Comparisons of biological parameters (engorged female weight, egg mass weight, and female-to-egg weight conversion efficiency index) indicated significant differences between different genotypes. The differences appeared to be heritable, but not related to amitraz resistance. Results from this study may have significant implications for the management of amitraz resistance.     

Ultrastructural alterations to chloroplasts in triazine-resistant weed biotypes

Ultrastructural, morphometric and physiological techniques were used to determine the consistent chloroplast differences between triazine-resistant (R) and triazine-susceptible (S) biotypes of Amaranthus hybridus L., Chenopodium album L., and Brassica campestris L. All R biotypes had a larger proportion of the chloroplast volume as grana lamellae and a lower proportion of starch and stroma lamellae than S biotypes. In the R biotypes, a greater percentage of grana contain larger numbers of thylakoids per granum. A greater proportion of chlorophyll associated with the light-harvesting chlorophyll alb protein and a lower chlorophyll alb ratio, traits associated with an increase in grana lamellae, were noted in R biotypes. Chloroplasts of S biotypes could be modified to ultrastructural phenocopies of those in R biotypes by treatment with sublethal levels of the PSII inhibiting herbicides, bentazon, diuron, atrazine and prometon. Despite the structural similarities to R biotypes, the modified S biotypes were not resistant to atrazine as determined by fluorescence measurements. Thus, the structural alterations observed are apparently secondary effects of impaired photosynthetic electron transport in R biotypes, and are not the cause of triazine resistance.     

Identification of tenuazonic acid as a novel type of natural photosystem II inhibitor binding in QB-site of Chlamydomonas reinhardtii

Tenuazonic acid (TeA) is a natural phytotoxin produced by Alternaria alternata, the causal agent of brown leaf spot disease of Eupatorium adenophorum. Results from chlorophyll fluorescence revealed TeA can block electron flow from Q_A to Q_B at photosystem II acceptor side. Based on studies with D1-mutants of Chlamydomonas reinhardtii, the No. 256 amino acid plays a key role in TeA binding to the Q_B-niche. The results of competitive replacement with [¹⁴C]atrazine combined with JIP-test and D1-mutant showed that TeA should be considered as a new type of photosystem II inhibitor because it has a different binding behavior within Q_B-niche from other known photosystem II inhibitors. Bioassay of TeA and its analogues indicated 3-acyl-5-alkyltetramic and even tetramic acid compounds may represent a new structural framework for photosynthetic inhibitors.     

Characterization of the alterations of the chlorophyll a fluorescence induction curve after addition of Photosystem II inhibiting herbicides

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, 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. This revised version was published online in August 2006 with corrections to the Cover Date.     

Photoconsumption of oxygen in photosystem II preparations under impairment of the water-oxidizing complex

Oxygen consumption in photosystem II (PSII) preparations in the light was 2 mol O₂/h per mg Chl at weakly acidic and at neutral pH values. It increased fourfold to fivefold at pH 8.5-9.0. The addition of either artificial electron donors for PSII such as MnCl₂ or diphenylcarbazide, or diuron as an inhibitor of electron transfer from Q_A, the primary bound quinone acceptor, to Q_B, the secondary bound quinone acceptor of PSII, resulted in a decrease in oxygen consumption rate at basic pH to value close to ones measured at pH 6.5. Such additions did not affect oxygen consumption at lower pH values. The induction of variable chlorophyll fluorescence yield in the light differed greatly at pH 6.5 and 8.5. While at pH 6.5 the fluorescence yield, after an initial fast rise almost to F_max, only slightly decreased, at pH 8.5 after such a rise it dropped promptly to a low value. The additions of the artificial electron donors at pH 8.5 resulted in the induction kinetics close to that observed at pH 6.5. These data indicate impairment of electron donation to P680⁺ that could be caused by damage to the water oxidation system at basic pH values. In experiments with PSII preparations treated with Tris to destroy the water-oxidizing complex, photoconsumption of oxygen in the entire pH region was close to the values in untreated preparations at basic pH. In untreated preparations the rate of light-induced oxygen consumption decreased in the presence of catalase, which decomposes H₂O₂, as well as in the presence of electron acceptor potassium ferricyanide. From these data it is suggested that the light-induced oxygen consumption in PSII is caused by two processes, by an interaction of O₂ with organic radicals, which were formed due to oxidation of components of the donor side of this photosystem (proteins, lipids, pigments) by cation-radical P680⁺, as well as by oxygen reduction by still unidentified components of PSII.     

Short-term responses of Photosystem I to heat stress

When 23°C-grown potato leaves (Solanum tuberosum L.) were exposed for 15 min to elevated temperatures in weak light, a dramatic and preferential inactivation of Photosystem (PS) II was observed at temperatures higher than about 38°C. In vivo photoacoustic measurements indicated that, concomitantly with the loss of PS II activity, heat stress induced a marked gas-uptake activity both in far-red light (>715 nm) exciting only PS I and in broadband light (350–600 nm) exciting PS I and PS II. In view of its suppression by nitrogen gas and oxygen and its stimulation by high carbon-dioxide concentrations, the bulk of the photoacoustically measured gas uptake by heat-stressed leaves was ascribed to rapid carbon-dioxide solubilization in response to light-modulated stroma alkalization coupled to PS I-driven electron transport. Heat-induced gas uptake was observed to be insensitive to the PS II inhibitor diuron, sensitive to the plastocyanin inhibitor HgCl₂ and saturated at a rather high photon flux density of around 1200 E m^–2 s^–1. Upon transition from far-red light to darkness, the oxidized reaction center P700⁺ of PS I was re-reduced very slowly in control leaves (with a half time t_1/2 higher than 500 ms), as measured by leaf absorbance changes at around 820 nm. Heat stress caused a spectacular acceleration of the postillumination P700⁺ reduction, with t_1/2 falling to a value lower than 50 ms (after leaf exposure to 48°C). The decreased t_1/2 was sensitive to HgCl₂ and insensitive to diuron, methyl viologen (an electron acceptor of PS I competing with the endogenous acceptor ferredoxin) and anaerobiosis. This acceleration of the P700⁺ reduction was very rapidly induced by heat treatment (within less than 5 min) and persisted even after prolonged irradiation of the leaves with far-red light. After heat stress, the plastoquinone pool exhibited reduction in darkness as indicated by the increase in the apparent Fo level of chlorophyll fluorescence which could be quenched by far-red light. Application (for 1 min) of far-red light to heat-pretreated leaves also induced a reversible quenching of the maximal fluorescence level Fm, suggesting formation of a pH gradient in far-red light. Taken together, the presented data indicate that PS I responded to the heat-induced loss of PS II photochemical activity by catalyzing an electron flow from stromal reductants. Heat-stress-induced PS I electron transport independent of PS II seems to constitute a protective mechanism since block of this electron pathway in anaerobiosis was observed to result in a dramatic photoinactivation of PS I.Abbreviations PFD photon flux density - PS Photosystem - Apt and Aox amplitude of the photothermal and photobaric components of the photoacoustic signal, respectively - P700 reaction center pigment of PS I - Fo and Fm initial and maximal levels of chlorophyll fluorescence, respectively - Fv=Fm Fo-variable chlorophyll fluorescence - Q_A primary (stable) electron acceptor of PS II - DCMU (diuron) 3-(3,4-dichlorophenyl)-1,1-dimethylurea - Cyt cytochrome     

Studies on the light-induced loss of the D1 protein in photosystem-II membrane fragments

PS II membrane fragments produced from higher plant thylakoids by Triton X-100 treatment exhibit strong photoinhibition and concomitant fast degradation of the D1 protein. Involvement of (molecular) oxygen is necessary for degradation of the D1 protein.The herbicides atrazine and diuron, but not ioxynil, partly protect the D1 protein against degradation. Binding of atrazine to the D1 protein is necessary to protect the D1 polypeptide, as shown with PS II membrane fragments from an atrazine-resistant biotype of Chenopodium album which are protected by diuron not by atrazine.Abbreviations atrazine 2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine - Chl chlorophyll, diuron - (DCMU) 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DMBQ 2,5-dimethyl-p-benzoquinone - DCIP 2,6-dichlorophenol indophenol - DPC diphenylcarbazide - ioxynil 4-cyano-2,6-diiodophenol - k_b binding constant - Mes 4-morpholinoethanesulfonic acid - P-680 reaction-center chlorophyll a of photosystem-II - PAGE polyacrylamide gel electrophoresis - PS II photosystem-II - Q_A and Q_B primary and secondary quinone electron acceptors - Z electron donor to the photosystem-II reaction center - SDS sodium dodecylsulfate - Tricine N-2-hydroxy-1,1-bis(hydroxymethyl)ethylglycine