Photoconversion kinetics of chloroplast Photosystems I and II. Effect of Mg2+

The effect of divalent cations on the primary photoconversion kinetics of chloroplast Photosystems (PS) I and II was investigated by absorbance difference spectrophotometry in the ultraviolet (ΔA₃₂₀) and red (ΔA₇₀₀) regions and by fluorescence at room temperature. Three main chlorophyll (Chl) a fluorescence emission components were identified. Addition of 5 mM MgCl₂ to unstacked chloroplasts caused a 5–7-fold increase in F_vα, the variable fluorescence yield controlled by the α-centers. The fluorescence yield F_vβ controlled by the β-centers and the nonvariable fluorescence yield F₀ were only slightly changed by the treatment. The absolute number of α- and β-centers remained unchanged and independent of divalent cations. The rate constants K_α, K_β and K_P-700 determined from the photoconversion kinetics of Q_α, Q_β and P-700 were also unchanged by divalent cations, suggesting a constancy of the respective absorption cross-sections. Evidence is presented that the Mg²⁺ effect on Chl a fluorescence is not due simply to unstacking. Conclusion: (1) In the absence of divalent cations from the chloroplast suspending medium, the variable fluorescence yield is not complementary to the rate of PS II photochemistry. (2) A spillover of excitation from PS II to PS I in the absence of Mg²⁺ cannot account for the 7-fold lowering of the variable fluorescence yield F_vα at room temperature. The results are discussed in view of a model of excitation transfer and fluorescence emission in the pigment bed of PS II_α and PS II_β.     

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.     

Investigations on the electron transfer processes in Photosystem II: New insights by chlorophyll fluorescence decay measurements with additional saturating light pulses

Electron transfer processes in leaves were investigated by chlorophyll fluorescence decay measurements. A fast chlorophyll fluorescence decay was observed in the intact state, reflecting normal electron transfer in Photosystem II. After treatment with DCMU a slow chlorophyll fluorescence decay was measured due to blocked electron transfer after the primary quinone Q_A. Additional saturating light pulses, one between each two measuring pulses, were used to completely reduce Q_A of the intact leaf: the chlorophyll fluorescence decay became similar to that of a DCMU treated leaf. A decreased electron donation rate to the reaction centre P680 was obtained after treatment with hydroxylamine. The intensity of the additional saturating light pulses was not sufficient to reduce all Q_A under this condition and only a small increase of the average chlorophyll fluorescence decay time occurred. Following our previous paper [Berg et al. (1997) Photosynthetica 34, in press], we investigated the effects of water stress with the additional saturating light pulses. An almost complete reduction of Q_A was possible after water stress started. A small, but systematic shortening of the slow chlorophyll fluorescence decay followed, up to a relative loss of leaf mass of 80%. At this time a rapid shortening of the chlorophyll fluorescence decay occurred, caused by an electron deficiency at the donor site of PS II. Additional saturating light pulses had no effects on the chlorophyll fluorescence decay any more, revealing a radiationless recombination between the reduced primary quinone Q and the oxidized reaction centre P680⁺.     

Cobalt ions inhibit electron-transport activity of Photosystem II without affecting Photosystem I

Investigations on photosynthesis have greatly benefited by the use of specific inhibitors that affect a specific site of inhibition on the electron-transport chain. We show here for the first time that cobalt (Co²⁺) ions can be used specifically to inactivate electron donation to the reaction centre of Photosystem (PS) II without affecting PS I reactions. This conclusion is based on the following observations: (1) addition of exogenous electron donors such as NH₂OH does not relieve Co²⁺-induced inactivation of photoelectron transport or the lowering of steady-state chlorophyll a fluorescence yield; this suggests that the inhibition is beyond the NH₂OH donation site and before the fluorescence quencher Q, i.e., on the reaction centre complex itself. (2) Washing of Co²⁺-pretreated chloroplasts with isolation buffer to remove Co²⁺ does not relieve Co²⁺-induced inhibition of Hill activity, suggesting that the Co²⁺ effect is irreversible. (3) Co²⁺ did not alter the PS I reactions. Thus, Co²⁺-treated chloroplasts can be used to study PS I functions free from PS II reactions in isolated chloroplasts.     

Inhibition of photosynthetic oxygen evolution and electron transfer from the quinone acceptor Q<Subscript>A</Subscript><Superscript>−</Superscript> to Q<Subscript>B</Subscript> by iron deficiency

The effect of iron deficiency on photosynthetic electron transport in Photosystem II (PS II) was studied in leaves and thylakoid membranes of lettuce (Lactuca sativa, Romaine variety) plants. PS II electron transport was characterized by oxygen evolution and chlorophyll fluorescence parameters. Iron deficiency in the culture medium was shown to affect water oxidation and the advancement of the S-states. A decrease of maximal quantum yield of PS II and an increase of fluorescence intensity at step J and I of OJIP kinetics were also observed. Thermoluminescence measurements revealed that charge recombination between the quinone acceptor of PS II, Q_B, and the S₂ state of the Mn-cluster was strongly perturbed. Also the dark decay of Chl fluorescence after a single turnover white flash was greatly retarded indicating a slower rate of Q_A ⁻ reoxidation.     

Molecular mechanism of high-temperature-induced inhibition of acceptor side of Photosystem II

High-temperature-induced inhibition of the acceptor side of Photosystem II (PS II) was studied in tobacco thylakoids using oxygen evolution, chlorophyll a (Chl a) fluorescence and redox potential measurements. When thylakoids were heated at 2 °C/min from 25 to 50 °C, the oxygen evolving complex became inhibited between 32 and 45 °C, whereas the acceptor side of PS II tolerated higher temperatures. Variable Chl a fluorescence decreased more slowly than oxygen evolution, suggesting that transitions between some S-states occurred even after heat-induced inhibition of the oxygen evolving activity. 77 K emission spectroscopy reveals that heating does not cause detachment of the light-harvesting complex II from PS II, and thus the heat-induced increase in the initial F₀ fluorescence is due to loss of exciton trapping in the heated PS II centers. Redox titrations showed a heat-induced increase in the midpoint potential of the Q_A/Q_A ^-) couple from the control value of –80 mV to +40 mV at 50 °C, indicating a loss of the reducing power of Q_A ^-). When its driving force thus decreased, electron transfer from Q_A ^-) to Q_B in the PS II centers that still could reduce Q_A became gradually inhibited, as shown by measurements of the decay of Chl a fluorescence yield after a single turnover flash. Interestingly, the heat-induced loss of variable fluorescence and inhibition of electron transfer from Q_A ^-) to Q_B could be partially prevented by the presence of 5 mM bicarbonate during heating, suggesting that high temperatures cause release of the bicarbonate bound to PS II. We speculate that both the upshift in the redox potential of the Q_A/Q_A ^-) couple and the release of bicarbonate may be caused by a heat-induced structural change in the transmembrane D1 or D2 proteins. This structural change may, in turn, be caused by the inhibition of the oxygen evolving complex during heating.     

Effects of frost hardening, dehardening and freezing stress on in vivo chlorophyll fluorescence of seedlings of Scots pine (Pinus sylvestris L.)

Abstract. The kinetics of in vivo chlorophyll fluorescence of photosystem II (PS II) was measured at room temperature and 77 K during frost hardening of seedlings of Scots pine (Pinus sylvestris L.), and after exposure of frost-hardened shoots to sub-freezing temperatures. A more pronounced decrease in variable fluorescence yield for the upper exposed than for the lower shaded surface of the needles suggested that some photoinhibition occurred during prolonged frost hardening at 50 μmol photons m^?2 s^?1 and 4°C. Reversible inhibition of photosynthesis after exposure to sub-freezing temperatures was initially manifested as an increase of steady-state energy-dependent fluorescence quenching (q_E) and a reduction in the rate of O₂ evolution. Further inhibition after treatment at still lower temperatures caused a progressive decline of steady-state photochemical quenching (q_Q) and the rate of O₂ evolution, whereas q_E remained high. This implies an inactivation of enzymes in the photosynthetic carbon reduction cycle decreasing the consumption of ATP and NADPH, which is likely to cause an increase of membrane energization and a reduction of the primary electron acceptor (Q_A) of PS II. Alternatively, the changes in q_Q and q_E might be attributed to an inhibition of photophosphorylation. Severe, irreversible damage to photosynthesis resulted in a suppression of q_E and of variable fluorescence yield, probably because the photochemical efficiency of PS II was impaired. Changes in the fast fluorescence kinetics at room temperature after severe freezing damage were interpreted as an inhibition of the electron flow from Q_A to the plastoquinone pool. It is suggested that irreversible freezing injury to needles of frost-hardened P. sylvestris causes damage to the Q_B,-protein.     

D1:Glu244 and D1:Tyr246 of the bicarbonate-binding environment of Photosystem II moderate high light susceptibility and electron transfer through the quinone-Fe-acceptor complex

In cyanobacteria, Glu-244 and Tyr-246 of the Photosystem II (PS II) D1 protein are hydrogen bonded to two water molecules that are part of a hydrogen-bond network between the bicarbonate ligand to a non-heme iron and the cytosol. Ala substitutions were introduced in Synechocystis sp. PCC 6803 to investigate the roles of these residues and the hydrogen-bond network on electron transfer between the primary plastoquinone acceptor, Q_A, and the secondary plastoquinone acceptor, Q_B, of the quinone-Fe-acceptor complex. All mutants assembled PS II; however, an increase in the PS II to PS I ratio was apparent, particularly in the E244A:Y246A double mutant. The mutants also showed impaired oxygen evolution and retarded chlorophyll a fluorescence decays following single turnover actinic flashes, which appeared to be primarily due to reduced Q_B binding in the E244A strain and an enhanced back reaction with the S2 state of the oxygen-evolving complex in the Y246A mutant. Impaired PS II in the Y246A and E244A:Y246A mutants resulted in inactivation of the psbA gene encoding D1. The Y246A and E244A:Y246A mutants also showed high light sensitivity whereas the E244A mutant showed enhanced resilience towards photodamage. Unlike the control strain, all of the mutants were insensitive to the addition of formate or bicarbonate in assays following chlorophyll decay kinetics that reflect electron transfer between Q_A and Q_B, suggesting the bicarbonate binding environment was perturbed. Our data also indicate that waters W582 and W622 (PDB: 4UB6) have essential roles in maintaining the architecture of the acceptor side of PS II.     

Structural and functional organization of the photosystems in spinach chloroplasts. Antenna size,relative electron-transport capacity,and chlorophyll composition

The structural and functional organization of the spinach chloroplast photosystems (PS) I, II_α and II_β was investigated. Sensitive absorbance difference spectrophotometry in the ultraviolet (?A₃₂₀) and red (?A₇₀₀) regions of the spectrum provided information on the relative concentration of PS II and PS I reaction centers. The kinetic analysis of PS II and PS I photochemistry under continuous weak excitation provided information on the number (N) of chlorophyll (Chl) molecules transferring excitation energy to PS II_α, PS II_β and PS I. Spinach chloroplasts contained almost twice as many PS II reaction centers compared to PS I reaction centers. The number N_α of chlorophyll (Chl) molecules associated with PS II_α was 234, while N_β = 100 and N_{PS I} = 210. Thus, the functional photosynthetic unit size of PS II reaction centers was different from that of PS I reaction centers. The relative electron-transport capacity of PS II was significantly greater than that of PS I. Hence, under light-limiting green excitation when both Chl a and Chl b molecules are excited equally, the limiting factor in the overall electron-transfer reaction was the turnover of PS I. The Chl composition of PS I, PS II_α and PS II_β was analyzed on the basis of a core Chl a reaction center complex component and a Chl $\text{a}\text{b-}\text{LHC}$ component. There is a dissimilar Chl $\text{a}\text{b-}\text{LHC}$ composition in the three photosystems with 77% of total Chl b associated with PS II_α only. The results indicate that PS II_α, located in the membrane of the grana partition region, is poised to receive excitation from a wider spectral window than PS II_β and PS I.     

Dynamics of photosystem II heterogeneity in Dunaliella salina (green algae)

Based on the electron-transport properties on the reducing side of the reaction center, photosystem II (PS II) in green plants and algae occurs in two distinct forms. Centers with efficient electron-transport from Q_A to plastoquinone (Q_B-reducing) account for 75% of the total PS II in the thylakoid membrane. Centers that are photochemically competent but unable to transfer electrons from Q_A to Q_B (Q_B-nonreducing) account for the remaining 25% of total PS II and do not participate in plastoquinone reduction. In Dunaliella salina, the pool size of Q_B-nonreducing centers changes transiently when the light regime is perturbed during cell growth. In cells grown under moderate illumination intensity (500 E m^-2s^-1), dark incubation induces an increase (half-time 45 min) in the Q_B-nonreducing pool size from 25% to 35% of the total PS II. Subsequent illumination of these cells restores the steady-state concentration of Q_B-nonreducing centers to 25%. In cells grown under low illumination intensity (30 µE m^–2s^–1), dark incubation elicits no change in the relative concentration of Q_B-nonreducing centers. However, a transfer of low-light grown cells to moderate light induces a rapid (half-time 10 min) decrease in the Q_B-nonreducing pool size and a concomitant increase in the Q_B-reducing pool size. These and other results are explained in terms of a pool of Q_B-nonreducing centers existing in a steady-state relationship with Q_B-reducing centers and with a photochemically silent form of PS II in the thylakoid membrane of D. salina. It is proposed that Q_B-nonreducing centers are an intermediate stage in the process of damage and repair of PS II. It is further proposed that cells regulate the inflow and outflow of centers from the Q_B-nonreducing pool to maintain a constant pool size of Q_B-nonreducing centers in the thylakoid membrane.Abbreviations Chl chlorophyll - PS photosystem - Q_A primary quinone electron acceptor of PS II - Q_B secondary quinone electron acceptor of PS II - LHC light harvesting complex - F_o non-variable fluorescence yield - F_pl intermediate fluorescence yield plateau level - F_max maximum fluorescence yield - F_i mitial fluorescence yield increase from F_o to F_pl(F_pl-F_o) - F_v total variable fluorescence yield (F_max-F_o) - DCMU dichlorophenyl-dimethylurea     

High Yield Non-detergent Isolation of Photosystem I-Light-harvesting Chlorophyll II Membranes from Spinach Thylakoids: IMPLICATIONS FOR THE ORGANIZATION OF THE PS I ANTENNAE IN HIGHER PLANTS*

Styrene-maleic acid copolymer was used to effect a non-detergent partial solubilization of thylakoids from spinach. A high density membrane fraction, which was not solubilized by the copolymer, was isolated and was highly enriched in the Photosystem (PS) I-light-harvesting chlorophyll (LHC) II supercomplex and depleted of PS II, the cytochrome b₆/f complex, and ATP synthase. The LHC II associated with the supercomplex appeared to be energetically coupled to PS I based on 77 K fluorescence, P₇₀₀ photooxidation, and PS I electron transport light saturation experiments. The chlorophyll (Chl) a/b ratio of the PS I-LHC II membranes was 3.2 ± 0.9, indicating that on average, three LHC II trimers may associate with each PS I. The implication of these findings within the context of higher plant PS I antenna organization is discussed.     

Development of Photosystem II in dark grown Chlamydomonas reinhardtii. A light-dependent conversion of PS IIβ, QB-nonreducing centers to the PS IIα, QB-reducing form

The green alga Chlamydomonas reinhardtii is a facultative heterotroph and, when cultured in the presence of acetate, will synthesize chlorophyll (Chl) and photosystem (PS) components in the dark. Analysis of the thylakoid membrane composition and function in dark grown C. reinhardtii revealed that photochemically competent PS II complexes were synthesized and assembled in the thylakoid membrane. These PS II centers were impaired in the electron-transport reaction from the primary-quinone electron acceptor, Q_A, to the secondary-quinone electron acceptor, Q_B (Q_B-nonreducing centers). Both complements of the PS II Chl a–b light harvesting antenna (LHC II-inner and LHC II-peripheral) were synthesized and assembled in the thylakoid membrane of dark grown C. reinhardtii cells. However, the LHC II-peripheral was energetically uncoupled from the PS II reaction center. Thus, PS II units in dark grown cells had a -type Chl antenna size with only 130 Chl (a and b) molecules (by definition, PS II units lack LHC II-peripheral). Illumination of dark grown C. reinhardtii caused pronounced changes in the organization and function of PS II. With a half-time of about 30 min, PS II centers were converted froma Q_B-nonreducing form in the dark, to a Q_B-reducing form in the light. Concomitant with this change, PS II units were energetically coupled with the LHC II-peripheral complement in the thylakoid membrane and were converted to a PS II form. The functional antenna of the latter contained more than 250 Chl(a+b) molecules. The results are discussed in terms of a light-dependent activation of the Q_A-Q_B electron-transfer reaction which is followed by association of the PS II unit with a LHC II-peripheral antenna and by inclusion of the mature form of PS II (PS II) in the membrane of the grana partition region.Abbreviations Chl chlorophyll - PS photosystem - Q_A primary quinone electron acceptor of PS II - Q_B secondary quinone electron acceptor of PS II - LHC light harvesting complex - F₀ non-variable fluorescence yield - F_plf intermediate fluorescence yield plateau leyel - F_max maximum fluorescence yield - F_i initial fluorescence yield increase from F₀ to F_pl (F_pl–F₀) - F_v total variable fluorescence yield (F_m–F0) - DCMU dichlorophenyl-dimethylurea     

Light-harvesting chlorophyll a-b complex requirement for regulation of Photosystem II photochemistry by non-photochemical quenching

Recently, it has been suggested (Horton et al. 1992) that aggregation of the light-harvesting a-b complex (LHC II) in vitro reflects the processes which occur in vivo during fluorescence induction and related to the major non-photochemical quenching (qE). Therefore the requirement of this chlorophyll a-b containing protein complex to produce qN was investigated by comparison of two barley mutants either lacking (chlorina f2) or depressed (chlorina¹⁰⁴) in LHC II to the wild-type and pea leaves submitted to intermittent light (IL) and during their greening in continuous light. It was observed that qN was photoinduced in the absence of LHC II, i.e. in IL grown pea leaves and the barley mutants. Nevertheless, in these leaves qN had no (IL, peas) or little (barley mutants) inhibitory effect on the photochemical efficiency of Q_A reduction measured by flash dosage response curves of the chlorophyll fluorescence yield increase induced by a single turn-over flash During greening in continuous light of IL pea leaves, an inhibitory effect on Q_A photoreduction associated to qN developed as Photosystem II antenna size increased with LHC II synthesis. Utilizing data from the literature on connectivity between PS II units versus antenna size, the following hypothesis is put forward to explain the results summarized above. qN can occur in the core antenna or Reaction Center of a fraction of PS II units and these units will not exhibit variable fluorescence. Other PS II units are quenched indirectly through PS II-PS II exciton transfer which develops as the proportion of connected PS II units increases through LHC II synthesis.     

Picosecond fluorescence kinetics in spinach chloroplasts at room temperature. Effects of Mg2+

Single-photon timing with picosecond resolution is used to investigate the effect of Mg²⁺ on the room-temperature fluorescence decay kinetics in broken spinach chloroplasts. In agreement with an earlier paper (Haehnel, W., Nairn, J.A., Reisberg, P. and Sauer, K. (1982) Biochim. Biophys. Acta 680, 161–173), we find three components in the fluorescence decay both in the presence and in the absence of Mg²⁺. The behavior of these components is examined as a function of Mg²⁺ concentration at both the F₀ and the F_max fluorescence levels, and as a function of the excitation intensity for thylakoids from spinach chloroplasts isolated in the absence of added Mg²⁺. Analysis of the results indicates that the subsequent addition of Mg²⁺ has effects which occur at different levels of added cation. At low levels of Mg²⁺ (less than 0.75 mM), there appears to be a decrease in communication between Photosystem (PS) II and PS I, which amounts to a decrease in the spillover rate between PS II and PS I. At higher levels of Mg²⁺ (about 2 mM), there appears to be an increase in communication between PS II units and an increase in the effective absorption cross-section of PS II, probably both of these involving the chlorophyll $\text{a}\text{b}$ light-harvesting antenna.     

Bicarbonate effect on electron flow in a cyanobacteriumSynechocystis PCC 6803

In this communication, evidence is presented from the kinetics of Q_A ^? decay (where Q_A is the first plastoquinone electron acceptor of photosystem II) and oxygen evolution for the requirement of bicarbonate in the electron transport in a cyanobacteriumSynechocystis (Pasteur Culture Collection 6803). A large slowing down of Q_A ^? oxidation, measured from the variable chlorophylla fluorescence after saturating actinic flashes, was observed in the thylakoids ofSynechocystis 6803 depleted of bicarbonate in the presence of 25 mM formate. Qualitatively similar results were obtained with DCMU-treated thylakoids. This shows that bicarbonate depletion inhibits electron transport on the acceptor side of photosystem II between Q_A and the plastoquinone (PQ) pool in cyanobacteria. Addition of 2.5 mM HCO₃ ^? fully reversed the inhibition of electron flow caused by bicarbonate depletion. Two exponential phases of Q_A ^? decay, a fast one and a slow one, were observed with halftimes of approx. 400 μs (fast) and 26 ms (slow) at pH 6.5. At pH 7.5, these phases were approx. 330 μs (fast) and 21 ms (slow), respectively. The amplitude, but not the halftime, of the fast component decreased by about 70% (pH 6.5) or 50% (pH 7.5); this was accompanied by a concomittant increase in the slow phase. Twenty mM bicarbonate stimulated, by a factor of 4, the Hill reaction in bicarbonate-depletedSynechocystis cells. This effect is independent of CO₂ fixation as it was observed even in the presence of an inhibitor DBMIB.     

Purification of Photosystem II particles from Phormidium laminosum using the detergent dodecyl-β-d-maltoside. Properties of the purified complex

The purification and properties of a new oxygen-evolving Photosystem (PS) II particle from the thermophilic blue-green alga Phormidium laminosum are described. The activity of the lauryldimethylamine N-oxide PS II-enriched supernatant described previously (Stewart, A.C. and Bendall, D.S. (1979) FEBS Lett. 107, 308–312) was found to be stabilized for several days at 4°;C by the addition of a second detergent, dodecyl-β-d-maltoside (lauryl maltoside). The lauryl maltoside/lauryldimethylamine N-oxide extract could be fractionated by sucrose density gradient centrifugation. Very high rates of oxygen evolution, typically 1900–2400 μmol O₂/mg chlorophyll a per h at pH 7 with dimethylbenzoquinone and ferricyanide as acceptors, were observed for the lowest green band from the gradient. This fraction contained cytochromes b-559 (high-potential) and c-549, but was completely devoid of P-700 and cytochromes b-563 and f. The purified oxygen-evolving particles comprised seven major polypeptides (M_r 58 900, 52 400, 43 200, 33 900, 30 000, 16 000 and 15 000) and approximately five minor polypeptides. The particles contained 3–4 Mn atoms per reaction centre and had a chlorophyll antenna of approx. 50 chlorophyll a. The fast phase of fluorescence induction curves in the presence of hydroxylamine and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) could be described by an exponential, suggesting that no energy transfer was occurring between the PS II units responsible for this phase. Comparison of the area above the fluorescence induction curves in the absence and presence of DCMU suggested an acceptor pool size of 2–3 equivalents per centre.     

Novel effects of methyl viologen on photosystem II function in spinach leaves

Methyl viologen (MV) is a well-known electron mediator that works on the acceptor side of photosystem I. We investigated the little-known, MV-induced inhibition of linear electron flow through photosystem II (PS II) in spinach-leaf discs. Even a low [MV] decreased the (1) average, light-adapted photochemical efficiency of PS II traps, (2) oxidation state of the primary quinone acceptor Q_A in PS II during illumination, (3) photochemical efficiency of light-adapted open PS II traps, (4) fraction of absorbed light energy dissipated constitutively in a light-independent manner or as chlorophyll (Chl) a fluorescence emission, (5) Chl a fluorescence yield corresponding to dark-adapted open reaction-center traps (F _o) and closed reaction-center traps (F _m), and (6) half-time for re-oxidation of Q_A⁻ in PS II after a single-turnover flash. These effects suggest that the presence of MV accelerates various “downhill” electron-transfer steps in PS II. Therefore, when using the MV to quantify cyclic electron flow, the inhibitory effect of MV on PS II should be taken into account.     

Excitation spectra of chlorophyll fluorescence in spinach and barley chloroplasts at 4 K

Excitation spectra of chlorophyll a fluorescence in chloroplasts from spinach and barley were measured at 4.2 K. The spectra showed about the same resolution as the corresponding absorption spectra. Excitation spectra for long-wave chlorophyll a emission (738 or 733 nm) indicate that the main absorption maximum of the photosystem (PS) I complex is at 680 nm, with minor bands at longer wavelengths. From the corresponding excitation spectra it was concluded that the emission bands at 686 and 695 nm both originate from the PS II complex. The main absorption bands of this complex were at 676 and 684 nm. The PS I and PS II excitation spectra both showed a contribution by the light-harvesting chlorophyll $\text{a}\text{b}$ protein(s), but direct energy transfer from PS II to PS I was not observed at 4 K. Omission of Mg²⁺ from the suspension favored energy transfer from the light-harvesting protein to PS I. Excitation spectra of a chlorophyll b-less mutant of barley showed an average efficiency of 50–60% for energy transfer from β-carotene to chlorophyll a in the PS I and in the PS II complexes.     

The effect of redox potential on the kinetics of fluorescence induction in Photosystem II particles from Phormidium laminosum. Sigmoidicity,energy transfer and the slow phase

Fluorescence induction curves in 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-inhibited Photosystem (PS) II particles isolated from the blue-green alga Phormidium laminosum have been analysed as a function of redox potential. Redox titration of the initial fluorescence indicated a single component with E_m,7.5 = +30 mV (n = 1) (Bowes, J., Horton, P. and Bendall, D.S. (1981) FEBS Lett. 135, 261–264). Despite this simplified electron acceptor system and the small number of chlorophylls per reaction centre, a sigmoidal induction curve was nevertheless seen. Sigmoidicity decreased as Q was reduced potentiometrically prior to induction such that the induction was exponential when the ratio $\text{F}{}_{\mathrm{<mtext>i</mtext>}}\text{F}{}_{\mathrm{<mtext>m</mtext>}}\text{= 0.64}$. These particles also showed a slow (β) phase of induction which titrated with an E_m value slightly more positive than that of the major quencher. It is concluded that the sigmoidal shape of the fluorescence induction curve observed in Phormidium PS II particles is not a consequence of a requirement for two photons to close the PS II reaction centre, but is generated as a result of energy transfer between photosynthetic units comprising one reaction centre per approx. 50 chlorophylls. Also, the existence of PS II heterogeneity (PS II_α, PS II_β centres) does not require a structurally differentiated chloroplast, but may only indicate the extent of aggregation of PS II centres.     

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