Kok's oxygen clock: What makes it tick? The structure of P680 and consequences of its oxidizing power

New insights in the structure of P680, the primary electron donor in Photosystem II, are summarized and the implications of its oxidizing power for energy transfer and singlet oxygen production are discussed.Abbreviations BChl bacteriochlorophyll - Chl chlorophyll - LD linear dichroism - Pheo pheophytin - PS II Photosystem II     

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

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

Characterization of the Carotenoidless Strain of Scenedesmus obliquus,Mutant C-6E,a Living Photosystem I Model*

When grown heterotrophically in the dark on enriched culture medium, the pigment-deficient strain of Scenedesmus obliquus, mutant C-6E, is uniquely characterized by a complete deficiency in carotenoids and chlorophyll b while retaining a low level of chlorophyll a which is exclusively utilized in photosystem I-type reactions. The strain lacks photosystem II activity but exhibits all PS-I reactions tested, including P700 redox reactions, photoreduction of CO₂ with hydrogen as electron donor, and O₂ uptake following methyl viologen reduction. The mutant contains 10 times more P700 per chlorophyll than the wild type and develops the pigment-protein complex of PS-I, CP-I. The action spectrum for methyl viologen reduction compares favorable to the low temperature absorption spectrum of whole cells. Both the chlorophyll fluorescence excitation and emission spectra of pigment-protein complexes derived from cells of C-6E show patterns typical of PS-I. The strain lacks the LHCs and CP-II as well as their respective apoproteins. The absence of carotenoids appears to prevent the development of the normal variety of pigment-protein complexes and the accumulation of Chl b. This inability is also expressed by the presence of only single stranded thylakoid membranes in the chloroplast of C-6E. When heterotrophically grown cells of this mutant are exposed to white light of 8 or 22 W m^?2, 50% of its chlorophyll is lost by photooxidation within 4 or 1.5 hours, respectively.     

胡杨、灰叶胡杨光合及叶绿素荧光特性的比较研究

比较研究了两年生胡杨和灰叶胡杨叶片的光合及叶绿素荧光特性.结果表明:胡杨和灰叶胡杨均表现出净光合速率(Pn)日变化呈单峰曲线,只是在12:00时胡杨的Pn略微降低;气孔导度日变化均呈单峰型曲线;胞间CO2浓度日变化呈近“V”字型曲线.相同的试验条件下,胡杨的净光合速率、气孔导度高于灰叶胡杨,净光合速率与气孔导度峰值出现在上午10:00时;胡杨胞间CO2浓度低于灰叶胡杨,胞间CO2浓度最低值均出现在中午14:00时.经充分暗适应后的叶片叶绿素荧光参数初始荧光、PSⅡ原初光能转换效率和PSⅡ潜在活性均为胡杨显著大于灰叶胡杨.以太阳光为光化学光,测定的叶绿素荧光参数日变化显示,胡杨PSⅡ实际的光化学反应量子效率、非循环电子传递速率、光化学猝灭系数均大于灰叶胡杨,而非光化学猝灭系数却较灰叶胡杨小.胡杨与灰叶胡杨在光合与叶绿素荧光特性上的差异,是胡杨更能适应干旱荒漠区高光、高温与低相对空气湿度环境,从而表现出高净光合速率的部分生理学原因之一.     

Calculation of absolute photosystem I absorption cross-sections from P700 photo-oxidation kinetics

A procedure is described which permits determination of the absolute absorption cross-section of a photosynthetic unit from the kinetics of reaction center photo-oxidation under weak, continuous actinic illumination. The method was first tested on a simple model compound of known absorption cross-section. We then applied the technique to absorption cross-section and functional antenna size measurements in photosystem I (PS I). A kinetic model is presented that can be used to fit P700 photo-oxidation measurements and extract the effective photochemical rate constant. The procedure is shown to properly correct for sample scattering and for the presence of heterogeneous absorbers (pigments not functionally coupled to P700). The relevance of these corrections to comparisons of antenna size using techniques that measure relative absorption cross-sections is discussed. Measurements on pea thylakoids in the presence and absence of 5 mM MgCl₂ show a 45% increase in PS I absorption cross-section in unstacked thylakoids. Analysis of detergent-isolated native PS I preparations (200 chlorophyll a+b/P700) clearly indicate that the preparation contains a broad distribution of antenna sizes. Finally, we confirm that Chlamydomonas reinhardtii strain LM3-A4d contains a PS I core antenna complex which binds only 60 chlorophyll a/P700, about half the functional size of the wild type complex. Limitations associated with calculation of functional antenna size from cross-section measurements are also discussed.Abbreviations PS photosystem - PS I-200 detergent-isolated photosystem I preparation containing about 200 Chl a+b/P700 - A _xxx absorbance at xxx nm - absolute absorption cross-section - I _a rate of light absorption - In _o incident actinic light intensity - _p quantum yield of photochemistry - k _eff effective rate constant for P700 photo-oxidation measured under conditions of limiting actinic intensity - k _r rate constant for P700⁺ reduction     

Modification of electron transfer from the quinone electron carrier,A1, of Photosystem 1 in a site directed mutant D576→L within the Fe-Sx binding site of PsaA and in second site suppressors of the mutation in Chlamydomonas reinhardtii

A site directed mutant of the Photosystem I reaction center of Chlamydomonas reinhardtii has been described previously. [Hallahan et al. (1995) Photosynth Res 46: 257–264]. The mutation, PsaA: D576L, changes the conserved aspartate residue adjacent to one of the cysteine ligands binding the Fe-S_X center to PsaA. The mutation, which prevents photosynthetic growth, was observed to change the EPR spectrum of the Fe-S_A/B centers bound to the PsaC subunit. We suggested that changes in binding of PsaC to the PsaA/PsaB reaction center prevented efficient electron transfer. Second site suppressors of the mutation have now been isolated which have recovered the ability to grow photosynthetically. DNA analysis of four suppressor strains showed the original D576L mutation is intact, and that no mutations are present elsewhere within the Fe-S_X binding region of either PsaA or PsaB, nor within PsaC or PsaJ. Subsequent genetic analysis has indicated that the suppressor mutation(s) is nuclear encoded. The suppressors retain the altered binding of PsaC, indicating that this change is not the cause of failure to grow photosynthetically. Further analysis showed that the rate of electron transfer from the quinone electron carrier A₁ to Fe-S_X is slowed in the mutant (by a factor of approximately two) and restored to wild type rates in the suppressors. ENDOR spectra of A₁ ^·– in wild-type and mutant preparations are identical, indicating that the electronic structure of the phyllosemiquinone is not changed. The results suggest that the quinone to Fe-S_X center electron transfer is sensitive to the structure of the iron-sulfur center, and may be a critical step in the energy conversion process. They also indicate that the structure of the reaction center may be modified as a result of changes in proteins outside the core of the reaction center.     

Effects of selective destruction of iron-sulfur center B on electron transfer and charge recombination in Photosystem I

Incubation of spinach thylakoids with HgCl₂ selectively destroys Fe–S center B (F_B). The function of electron acceptors in F_B-less PS I particles was studied by following the decay kinetics of P700⁺ at room temperature after multiple flash excitation in the absence of a terminal electron acceptor. In untreated particles, the decay kinetics of the signal after the first and the second flashes were very similar (t _1/22.5 ms), and were principally determined by the concentration of the artificial electron donor added. The decay after the third flash was fast (t _1/20.25 ms). In F_B-less particles, although the decay after the first flash was slow, fast decay was observed already after the second flash. We conclude that in F_B-less particles, electron transfer can proceed normally at room temperature from F_X to F_A and that the charge recombination between P700⁺ and F_X ^-/A₁ ^- predominated after the second excitation. The rate of this recombination process is not significantly affected by the destruction of F_B. Even in the presence of 60% glycerol, F_B-less particles can transfer electrons to F_A at room temperature as efficiently as untreated particles.Abbreviations DCIP 2, 6-dichlorophenol indophenol - F_A, F_B, F_X iron-sulfur center A, B and X, respectively - PMS phenazine methosulfate     

Two electron donation sites for exogenous reductants in chloroplast Photosystem II

Two sites are distinguished for the oxidation of exogenous donors by Photosystem II in non-oxygen evolving chloroplasts. In the presence of lipophilic donors (e.g. phenylenediamine, benzidine, diphenylcarbazide), the rate for Signal IIf rereduction following a flash increases as the concentration of exogenous reductant increases. There is a decrease (20–40%) in Signal IIf magnitude accompanying donor addition at low (< 10^?5M) concentrations, but the extent of the decrease does not change further with increasing donor concentration. Complementary polarographic experiments monitoring donor (phenylenediamine) oxidation show an increase in oxidation rate with increasing donor concentration.In the presence of the hydrophilic donor, Mn²⁺, the Signal IIf decay halftime remains constant with increasing Mn²⁺ concentration. However, the flash-induced Signal IIf magnitude progressively decreases with increasing Mn²⁺ concentration.These results are interpreted in terms of two competing paths for the reduction of P680⁺. In one path P680⁺ reduction is accompanied by the appearance of Signal IIf, and lipophilic donors subsequently rereduce the Signal IIf species in a series reaction. This reduction follows pseudo-first order kinetics as a function of donor concentration. In the second path Mn²⁺ reduces P680⁺ in a parallel reaction that competes with the formation of the Signal IIf species. This results in a decrease in the magnitude of Signal IIf, but no change in its decay time.     

Evaluation of the Effect of Light Intensity on the Measurement of the Photosynthetic Rate in Plankton Microalgae by the Chlorophyll Fluorescence Method

The use of relative variable fluorescence (RVF) of chlorophyll, as measured in the presence of Diuron, an inhibitor of electron transfer, for the estimation of the photosynthetic activity of plankton microalgae was analyzed under a wide range of light intensities in the PAR region. Oxygen evolution rates (estimated by the method of light and dark bottles and the amperometric method), RVF, and chlorophyll a concentration were measured in parallel in natural algal cenoses and microecosystems. When the previously used regression equation, in the form A = b(F/F_d)C_chlI, where A is O₂ evolution rate (g/(m³ h), F/F_d is RVF (relative units), C_chl is chlorophyll a concentration (mg/m³), and I is light intensity (W/m²), was verified in the PAR region, we observed a nonlinear dependence of the correction coefficient b on I, which can be described by the formula b = 6.227 × 10³I. This result agrees with the hypothesis that chlorophyll a fluorescence quenching comprises photochemical (qQ) and energy (qE) components. On the basis of the energy model, we determined the upper limit b_max = 0.003 for light intensity range I< 4.4 W/m² and the lower limit b_min = 0.0003 for I = 400 W/m².     

Characteristics of Photosystem I Complexes in the End Membranes of Pea Granal Thylakoids

Two fractions of the light fragments enriched in the photosystem I (PSI) complexes were obtained from pea (Pisum sativum L.) thylakoids by digitonin treatment and subsequent differential centrifugation. The ratio of chlorophyll a to chlorophyll b, chlorophyll/P700 spectra of low-temperature fluorescence, and excitation spectra of long-wave fluorescence were measured. These characteristics were shown to be different due to variation in the size and composition of the light-harvesting antenna of PSI complexes present in the particles obtained. The larger antenna size of one of the fractions was related to the incorporation of the pool of light-harvesting complex II (LHCII). A comparison with the data available allowed us to identify these particles as fragments of intergranal thylakoids and end membranes of granal thylakoids. The suggestion that an increase in the PSI light-harvesting antenna in intergranal thylakoids is related to the attachment of phosphorylated LHCII is discussed.     

Factors Limiting Photosynthetic Recovery in Sweet Pepper Leaves After Short-Term Chilling Stress Under Low Irradiance

The effects of chilling treatment (4 °C) under low irradiance, LI (100 mol m^–2 s^–1) and in the dark on subsequent recovery of photosynthesis in chilling-sensitive sweet pepper leaves were investigated by comparing the ratio of quantum yields of photosystem (PS) 2 and CO₂ assimilation, _PS2/_CO2, measured in normal air (21 % O₂, NA) and low O₂-air (2% O₂, LOA), and by analyzing chlorophyll (Chl) a fluorescence parameters. Chilling treatment in the dark had little effect on F_v/F_m and _PS2/_CO2, but it caused the decrease of net photosynthetic rate (P _N) under saturating irradiance after 6-h chilling treatment, indicating that short-term chilling alone did not induce PS2 photoinhibition. Furthermore, photorespiration and Mehler reaction also did not obviously change during subsequent recovery after chilling stress in the dark. During chilling treatment under LI, there were obvious changes in F_v/F_m and _PS2/_CO2, determined in NA or LOA. F_v/F_m could recover fully in 4 h at 25 °C, and _PS2/_CO2 increased at the end of the treatment, as determined in both NA and LOA. During subsequent recovery, _PS2/_CO2 in LOA decreased faster than in NA. Thus the Mehler reaction might play an important role during chilling treatment under LI, and photorespiration was an important process during the subsequent recovery. The recovery of PN under saturating irradiance determined in NA and LOA took about 50 h, implying that there were some factors besides CO₂ assimilation limiting the recovery of photosynthesis. From the progress of reduced P700 and the increase of the Mehler reaction during chilling under LI we propose that active oxygen species were the factors inducing PS1 photoinhibition, which prevented the recovery of photosynthesis in optimal conditions because of the slow recovery of the oxidizable P700.     

Photosynthesis in different types of transgenic tobacco plants with elevated cytokinin content

Photosynthetic parameters were compared in three types of transgenic tobacco plants: ipt-transgenic plants with slightly elevated endogenous cytokinin (CK) content, Pssu-ipt-transgenic plants with markedly increased CK content, and zmp-transgenic plants with slightly elevated CK content accompanied by elevated auxin content. Slightly increased CK content promoted net photosynthetic rate (P_N) in both ipt- and zmp-transgenic plants, and chlorophyll (Chl) and carotenoid contents in zmp-transgenic plants. Morphology, growth characteristics, stomatal conductance, and parameters of Chl a fluorescence kinetics were similar in control and transgenic plants with slightly higher CK content. No significant effect of increased level of endogenous auxin (indole-3-acetic acid) on development of zmp-transgenic plants and measured parameters was found. Pssu-ipt-transgenic plants with highly increased CK content revealed suppressed root development, wilting of plants, and depression of P_N and stomatal conductance; however, Chl content was slightly increased and parameters of Chl a fluorescence kinetics did not indicate damage to photosynthetic apparatus. This revised version was published online in July 2006 with corrections to the Cover Date.     

Responses of photosystem I compared with photosystem II to high-light stress in tropical shade and sun leaves

Sun and shade leaves of several plant species from a neotropical forest were exposed to excessive light to evaluate the responses of photosystem I in comparison to those of photosystem II. Potential photosystem I activity was determined by means of the maximum P700 absorbance change around 810 nm (ΔA_810max) in saturating far-red light. Leaf absorbance changes in dependence of increasing far-red light fluence rates were used to calculate a ‘saturation constant’, K_s, representing the far-red irradiance at which half of the maximal absorbance change (ΔA_810max/2) was reached in the steady state. Photosystem II efficiency was assessed by measuring the ratio of variable to maximum chlorophyll fluorescence, F_v/F_m, in dark-adapted leaf samples. Strong illumination caused a high degree of photo-inhibition of photosystem II in all leaves, particularly in shade leaves. Exposure to 1800–2000 μ mol photons m⁻² s⁻¹ for 75 min did not substantially affect the potential activity of photosystem I in all species tested, but caused a more than 40-fold increase of K_s in shade leaves, and a three-fold increase of K_s in sun leaves. The increase in K_s was reversible during recovery under low light, and the recovery process was much faster in sun than in shade leaves. The novel effect of high-light stress on the light saturation of P700 oxidation described here may represent a complex reversible mechanism within photosystem I that regulates light-energy dissipation and thus protects photosystem I from photo-oxidative damage. Moreover, we show that under high-light stress a high proportion of P700 accumulates in the oxidized state, P700⁺. Presumably, conversion of excitation energy to heat by this cation radical may efficiently contribute to photoprotection.     

<Emphasis Type="Italic">In vivo</Emphasis> carotenoid triplet formation in response to excess light: a supramolecular photoprotection mechanism revisited

Carotenoids have been known for their photoprotective role for about 50 years. However, despite many advances in laser flash photolysis, no photodynamic studies have been so far performed on whole cells to determine the harmful threshold of light. In the present work, we investigate the coupling between energy conversion and energy deactivation, in isolated complexes of RC-LH1 and LH2 increasingly integrated systems up to intact cells of the purple anaerobic photosynthetic bacterium Rubrivivax gelatinosus. A continuous light similar to the mean daily sun irradiance on the surface of the earth is found to saturate the in vivo electron transfer turnover and to give rise to carotenoid triplet formation. This accounts for the widespread use of carotenoids among phototrophic prokaryotes and emphasizes their essential protective role in the natural environment.     

Influence of in vitro growth conditions on photosynthetic competence and survival rate of Rehmannia glutinosa plantlets during acclimatization period

The photosynthetic responses of Rehmannia glutinosa grown under photoautotrophic or heterotrophic conditions in vitro were investigated after transfer to greenhouse conditions. In addition, the changes in carbohydrate content and survival rates of the plantlets were evaluated. During six days after transplantation, the photosynthetic rate declined and photoinhibitory impairments represented by decrease of Fv/Fm and chlorophyll content were observed regardless of environmental conditions in vitro. Excessive transpiration was observed in plantlets grown under heterotrophic conditions during that period. Fructose and glucose content of the plantlets grown under photoautotrophic conditions increased with time and reached almost the same level of field grown plants after day 15. Under heterotrophic conditions, in contrast, the content of these sugars decreased continuously during that period. It is suggested that high survival rate of plantlets grown under photoautotrophic conditions has to be attributed to improvement of photosynthetic competence by imposed high light intensity and CO₂ concentration in vitro. The results strongly suggest that the control of transpiration during early stage after transplantation plays a key role in the acclimatization process, and photoautotrophic conditions could be a solution to solve the problems associated with transplantation stress. This revised version was published online in June 2006 with corrections to the Cover Date.     

Photosynthetic Units of Phototrophic Organisms

Photoautotrophic organisms play a key role in the biosphere of the Earth, converting solar energy of the 350-1000 nm range into biochemically available form. In contemporary aquatic and terrestrial ecosystems, the dominating groups are the oxygen evolving cyanobacteria, algae, and higher plants. Anoxygenic phototrophic microorganisms occupy mainly ecological niches with extreme environmental conditions. Despite diverse evolution of all these taxonomic groups, their photosynthetic apparatus has a similar molecular design and identical principles of operation. This review covers recent data about features of the structural and functional organization of pigment-protein complexes of the basic types of photosynthetic units in prokaryotes and eukaryotes. A correspondence between the optical properties of various photosynthetic units and the natural light conditions is discussed.