Effects of ambient versus reduced UV-B radiation on high arctic Salix arctica assessed by measurements and calculations of chlorophyll a fluorescence parameters from fluorescence transients

A UV-B exclusion-experiment was conducted in the high Arctic Zackenberg, NE Greenland, in which Salix arctica leaves during most of the growing season were fixed perpendicular to the solar zenith angle, thereby receiving maximal solar radiation. Covered with Teflon and Mylar foil, the leaves received approximately 90 and 40% of the ambient UV-B irradiance, respectively. The effects were examined through recordings of chlorophyll a fluorescence transients, determination of biomass and analysis of total carbon and nitrogen content and amount of soluble flavonoids in the leaves. The processing of light was analysed by means of the chlorophyll a fluorescence transient, using the so-called JIP test, as evolved by Reto J. Strasser and his coworkers. Reduction of the UV-B irradiance caused a rise in many of the fluorescence parameters during July, but not in August (late season). Thus increases in the efficiency that an absorbed photon will be trapped by the PSII reaction centre with the resultant reduction of Q_A to Q_A^– (ET₀/ABS = F_V/F_M) and the efficiency that an electron residing on Q_A^– will enter the intersystem electron transport chain (ET₀/TR₀) were observed in reduced UV-B. Moreover, estimated per cross-section of leaf sample, the number of active PSII reaction centres (RC/CS_M) and electron transport rate (ET_M/CS_M) and all performance indexes (PI_ABS, PI_CSo and PI_CSm) were increased in reduced UV-B. The total soluble flavonoid content was highest in ambient UV-B. The treatment effects on fluorescence parameters that were directly measured (e.g. F₀ and F_M) and those that were derived (e.g. quantum efficiencies, parameters per PSII reaction centres and per cross-section of leaf sample) are discussed in relation to one another, in relation to daily and seasonal variation, and from the perspective of evaluating the relative importance of UV-B of donor and acceptor side capacity in Photosystem II. In conclusion, the experimental set-up and non-invasive measurements proved to be a sensitive method to screen for effects of UV-B stress.     

On the chlorophyll a fluorescence yield in chloroplasts upon excitation with twin turnover flashes (TTF) and high frequency flash trains

Chlorophyll fluorescence is routinely taken as a quantifiable measure of the redox state of the primary quinone acceptor Q_A of PSII. The variable fluorescence in thylakoids increases in a single turnover flash (STF) from its low dark level F _o towards a maximum F _m^STF when Q_A becomes reduced. We found, using twin single turnover flashes (TTFs) that the fluorescence increase induced by the first twin-partner is followed by a 20–30% increase when the second partner is applied within 20–100 μs after the first one. The amplitude of the twin response shows a period-of-four oscillation associated with the 4-step oxidation of water in the Kok cycle (S states) and originates from two different trapped states with a life time of 0.2–0.4 and 2–5 ms, respectively. The oscillation is supplemented with a binary oscillation associated with the two-electron gate mechanism at the PSII acceptor side. The F(t) response in high frequency flash trains (1–4 kHz) shows (i) in the first 3–4 flashes a transient overshoot 20–30% above the F _m^STF = 3*F _o level reached in the 1st flash with a partial decline towards a dip D in the next 2–3 ms, independent of the flash frequency, and (ii) a frequency independent rise to F _m = 5*F _o in the 3–60 ms time range. The initial overshoot is interpreted to be due to electron trapping in the S₀ fraction with Q_B-nonreducing centers and the dip to the subsequent recovery accompanying the reoxidation of the double reduced acceptor pair in these RCs after trapping. The rise after the overshoot is, in agreement with earlier findings, interpreted to indicate a photo-electrochemical control of the chlorophyll fluorescence yield of PSII. It is anticipated that the double exciton and electron trapping property of PSII is advantageous for the plant. It serves to alleviate the depression of electron transport in single reduced Q_B-nonreducing RCs, associated with electrochemically coupled proton transport, by an increased electron trapping efficiency in these centers.     

Recovery of photosystem I and II activities during re-hydration of lichen<Emphasis Type="Italic"> Hypogymnia physodes</Emphasis> thalli

Photochemical efficiencies of photosystem I (PSI) and photosystem II (PSII) were studied in dry thalli of the lichen Hypogymnia physodes and during their re-hydration. In dry thalli, PSII reaction centers are photochemically inactive, as evidenced by the absence of variable chlorophyll (Chl) fluorescence, whereas the primary electron donor of PSI, P700, exhibits irreversible oxidation under continuous light. Upon application of multiple- and, particularly, single-turnover pulses in dry lichen, P700 oxidation partially reversed, which indicated recombination between P700⁺ and the reduced acceptor F_X of PSI. Re-wetting of air-dried H. physodes initiated the gradual restoration of reversible light-induced redox reactions in both PSII and PSI, but the recovery was faster in PSI. Two slow components of P700⁺ reduction occurred after irradiation of partially and completely hydrated thalli with strong white light. In contrast, no slow component was found in the kinetics of re-oxidation of Q_A^–, the reduced primary acceptor of PSII, after exposure of such thalli to white light. This finding indicated the inability of PSII in H. physodes to provide the reduction of the plastoquinone pool to significant levels. It is concluded that slow alternative electron transport routes may contribute to the energetics of photosynthesis to a larger extent in H. physodes than in higher plants.Abbreviations A₀ and A₁ Primary acceptor chlorophyll and secondary electron acceptor phylloquinone - Chl a Chlorophyll a - F_m Maximal level of chlorophyll fluorescence when all PSII centers are closed - F_o Minimal level of fluorescence when all PSII centers are open after dark adaptation - FR Far-red - F_v Variable fluorescence (=F_m–F_o) - F_X, F_A, and F_B Iron–sulfur centers - MT pulse Multiple-turnover pulse - PS Photosystem - P700 Reaction center chlorophyll of PSI - Q_A Primary quinone acceptor of PSII - Q_B Secondary quinone acceptor of PSII - ST pulse Single-turnover pulse     

Photosystem II activity of wild type <Emphasis Type="Italic">Synechocystis</Emphasis> PCC 6803 and its mutants with different plastoquinone pool redox states

To assess the role of redox state of photosystem II (PSII) acceptor side electron carriers in PSII photochemical activity, we studied sub-millisecond fluorescence kinetics of the wild type Synechocystis PCC 6803 and its mutants with natural variability in the redox state of the plastoquinone (PQ) pool. In cyanobacteria, dark adaptation tends to reduce PQ pool and induce a shift of the cyanobacterial photosynthetic apparatus to State 2, whereas illumination oxidizes PQ pool, leading to State 1 (Mullineaux, C. W., and Holzwarth, A. R. (1990) FEBS Lett., 260, 245-248). We show here that dark-adapted Ox^– mutant with naturally reduced PQ is characterized by slower Q_A^– reoxidation and O₂ evolution rates, as well as lower quantum yield of PSII primary photochemical reactions (F_v/F_m) as compared to the wild type and SDH–mutant, in which the PQ pool remains oxidized in the dark. These results indicate a large portion of photochemically inactive PSII reaction centers in the Ox^– mutant after dark adaptation. While light adaptation increases F_v/F_m in all tested strains, indicating PSII activation, by far the greatest increase in F_v/F_m and O₂ evolution rates is observed in the Ox^– mutant. Continuous illumination of Ox^– mutant cells with low-intensity blue light, that accelerates Q_A^– reoxidation, also increases F_v/F_m and PSII functional absorption cross-section (590 nm); this effect is almost absent in the wild type and SDH–mutant. We believe that these changes are caused by the reorganization of the photosynthetic apparatus during transition from State 2 to State 1. We propose that two processes affect the PSII activity during changes of light conditions: 1) reversible inactivation of PSII, which is associated with the reduction of electron carriers on the PSII acceptor side in the dark, and 2) PSII activation under low light related to the increase in functional absorption cross-section at 590 nm.     

Photoinhibition and light-dependent turnover of the D1 reaction-centre polypeptide of photosystem II are enhanced by mineral-stress conditions

The function of photosystem II (PSII) and the turnover of its D1 reaction-center protein were studied in spinach (Spinacia oleracea L.) plants set under mineral stress. The mineral deficiencies were induced either by supplying the plants with an acidic nutrient solution or by strongly reducing the supply of magnesium alone or together with sulfur. After exposure for 8–10 weeks to the different media, the plants were characterized by a loss of chlorophyll and an increase in starch content, indicating a disturbance in the allocation of assimilates. Depending on the severity of the mineral deficiencies the plants lost their ability to adapt even to moderate iradiances of 400 mol photons·m^–2·s^–1 and became photoinhibited, as indicated by the decrease in F_v/F_m (the ratio of yield of variable fluorescence to yield of maximal fluorescence when all reaction centers are closed). The loss of PSII function was induced by changes on the acceptor side of PSII. Fast fluorescence decay showed a loss of PSII centers with bound Q_B, the secondary quinone acceptor of PSII, and a fast reoxidation kinetic of q _a ^- , the primary quinone acceptor of PSII, in the photoinactivated plants. No appreciable change could be observed in the amount of PSII centers with unbound Q_B and in Q_B-nonreducing PSII centers. Immunological studies showed that the contents of the D1 and D2 proteins of the PSII reaction center and of the 33-kDa protein of the water-splitting complex were diminished in the photoinhibited plants, and the occurrance of a new polypetide of 14 kDa that reacted with an antibody against the C-termius of the D1 protein. As shown by pulse-labelling experiments with [¹⁴C]leucine both degradation and synthesis of the D1 protein were enhanced in the mineral-deficient plants when compared to non-deficient plants. A stimulation of D1-protein turnover was also observed in pH 3-grown plants, which were not inhibited at growth-light conditions. Obviously, stimulation of D1-protein turnover prevented photoinhibition in these plants. However, in the Mg- and Mg/S-deficient plants even a further stimulation of D1-protein turnover could not counteract the increased rate of photoinactivation.Abbreviations amp_(f,m,s) amplitude of the fast, (medium and slow) exponential component of fluorescence decay - F_m yield of maximum fluorescenc when all reaction centers are closed - F_o yield of intrinsic fluorescence at open PSII reaction centers in the dark - F_v yield of variable fluorescence, (difference between F_m and F_o) - LHC light-harvesting complex - PFD photon flux density - Q_A primary quinone acceptor of PSII - Q_B secondary quinone acceptor of PSII Dedicated to Professor Dr. Dres. hc. Achim Trebst on the occasion of his 65th birthdayThis work was supported by grants from the BMFT and the Ministerium für Umwelt, Raumordnung and Landwirtschaft, Nordrhein-Westfalen. The authors thank H. Wietoska and M. Bronzel for skilful technical assistance.     

Resistance of reaction centers from Rhodobacter sphaeroides against UV-B radiation. Effects on protein structure and electron transport

Inhibition of electron transport and damage to the protein subunits by ultraviolet-B (UV-B, 280–320 nm) radiation have been studied in isolated reaction centers of the non-sulfur purple bacterium Rhodobacter sphaeroides R26. UV-B irradiation results in the inhibition of charge separation as detected by the loss of the initial amplitude of absorbance change at 430 nm reflecting the formation of the P⁺(Q_AQ_B)^– state. In addition to this effect, the charge recombination accelerates and the damping of the semiquinone oscillation increases in the UV-B irradiated reaction centers. A further effect of UV-B is a 2 fold increase in the half- inhibitory concentration of o-phenanthroline. Some damage to the protein subunits of the RC is also observed as a consequence of UV-B irradiation. This effect is manifested as loss of the L, M and H subunits on Coomassie stained gels, but not accompanied with specific degradation products. The damaging effects of UV-B radiation enhanced in reaction centers where the quinone was semireduced (Q_B ^–) during UV-B irradiation, but decreased in reaction centers which lacked quinone at the Q_B binding site. In comparison with Photosystem II of green plant photosynthesis, the bacterial reaction center shows about 40 times lower sensitivity to UV-B radiation concerning the activity loss and 10 times lower sensitivity concerning the extent of reaction center protein damage. It is concluded that the main effect of UV-B radiation in the purple bacterial reaction center occurs at the Q_AQ_B quinone acceptor complex by decreasing the binding affinity of Q_B and shifting the electron equilibration from Q_AQ_B ^– to Q_A ^–Q_B. The inhibitory effect is likely to be caused by modification of the protein environment around the Q_B binding pocket and mediated by the semiquinone form of Q_B. The UV-resistance of the bacterial reaction center compared to Photosystem II indicates that either the Q_AQ_B acceptor complex, which is present in both types of reaction centers with similar structure and function, is much less susceptible to UV damage in purple bacteria, or, more likely, that Photosystem II contains UV-B targets which are more sensitive than its quinone complex.Abbreviations Bchl bacteriochlorophyll - P Bchl dimer - Q_A primary quinone electron acceptor - Q_B secondary quinone electron acceptor - RC reaction center - UV-B ultraviolet-B     

Effect of ATP on the Content of Inactive PSII Complexes in Chlorella

A prolonged (20 h) dark incubation of Chlorella pyrenoidosa algae at 37°C resulted in a twofold rise of the slowly rising phase (10–15 min), sF _v, in the kinetics of variable chlorophyll fluorescence, F _v (F _v = F _m – F ₀) in diuron-treated cells. This effect suggests the accumulation of inactive photosystem II (PSII) complexes with low efficiency of primary quinone acceptor of electron of PSII (Q_A) reduction. The presence of methylamine (MA), a thylakoid membrane uncoupler, or N, N-dicyclohexylcarbodiimide, an inhibitor of ATPase, precluded the accumulation of inactive PSII complexes. When salicylhydroxamate promoted the reduction of the plastoquinone (PQ) pool, exogenous ATP accelerated the accumulation of inactive complexes. Dark PQ oxidation in the presence of nonmetabolized glucose analog, 2-deoxy-D-glucose, lowered the content of inactive PSII complexes, and NaF, an inhibitor of chloroplast phosphatases, retarded this process. These data are considered as evidence for a mechanism regulating the content of inactive PSII complexes in the process of redox-dependent phosphorylation of D1- and/or D2-proteins of PSII.     

Changes in D1-protein turnover and recovery of photosystem II activity precede accumulation of chlorophyll in plants after release from mineral stress

After seven weeks of a combined magnesium and sulphur deficiency, spinach (Spinacea oleracea L.) plants showed a substantial accumulation of inactivated photosystem II (PSII) centres as indicated by a 40% decrease of the chlorophyll (Chl) fluorescence parameter F_v/F_m (F_v being the yield of variable fluorescence and F_m the yield of maximal fluorescence when all reaction centres are closed) together with a severe loss of leaf Chl content of 75%. The responses of the photosynthetic apparatus were examined when the deficient plants were transferred back to a rich nutrient medium. During the first 24 h of the recovery phase, thylakoid protein synthesis measured as incorporation of [¹⁴C]leucine per unit of Chl increased substantially. The synthesis rate of the D1 reaction-centre polypeptide of PSII, which in the deficient plants was reduced to 50% of the non-deficient control, was stimulated eight- to ninefold. D1-protein content, which in the deficient plants was reduced to 40% of the non-deficient control, started to increase 2 d later. Thus, D1-protein degradation was also enhanced. The increased D1-protein turnover led to a rapid repair of the existing PSII centres as indicated by the rise of F_v/F_m. It was completed at day 7 of the recovery phase. At day 2 of the recovery phase, the synthesis of other thylakoid proteins such as the D2 protein, cytochrome b ₅₅₉, CP 47 and the 33-kDa polypeptide of the water-splitting system, became stimulated. This process resulted in an accumulation of new PSII centres. During the first week, formation of new PSII centres was not associated with an increase in leaf Chl content. The Chl content of the recovering leaves only started to increase when the ratio of PSII polypeptides versus LHCII (light-harvesting complex of PSII), which was substantially diminished in the deficient plants, became comparable to that of the control. The recovery process was accompanied by substantial changes in thylakoid protein phosphorylation. Their relevance to thylakoid protein turnover and stability is discussed.Abbreviations Chl chlorophyll - cyt cytochrome - F_o yield of intrinsic fluorescence when all PSII centres are open in the dark - F_m yield of maximal fluorescence when all reaction centres are closed - F_m fluorescence yield when all reaction centres are closed (after a saturating flash) under steady-state conditions - F_v yield of variable fluorescence, (difference between F_oand F_m) - F yield of variable fluorescence under steady state conditions - LHC light-harvesting complex - PQ plastoquinone - Q_A primary quinone acceptor of PSII - Q_B secondary quinone acceptor of PSII - q_P photochemical quenching - q_n non-photochemical quenching The authors like to thank Dipl. Biol. Britta Untereiser for determining the chlorophyll fluorescence quenching factors. This work was supported by grants from the Bundesminister für Forschung und Technologie, the Project Europäisches Forschungszentrum and the German Israeli Foundation in cooperation with Prof. I. Ohad, Hebrew University, Jerusalem, Israel.     

Analysis of the changes of electron transfer and heterogeneity of photosystem II in Deg1-reduced Arabidopsis plants

Deg1 protease functions in protease and chaperone of PSII complex components, but few works were performed to study the effects of Deg1 on electron transport activities on the donor and acceptor side of PSII and its correlation with the photoprotection of PSII during photoinhibition. Therefore, we performed systematic and comprehensive investigations of electron transfers on the donor and acceptor sides of photosystem II (PSII) in the Deg1-reduced transgenic lines deg1-2 and deg1-4. Both the maximal quantum efficiency of PSII photochemistry (F_v/F_m) and the actual PSII efficiency (Φ_PSII) decreased significantly in the transgenic plants. Increases in nonphotochemical quenching (NPQ) and the dissipated energy flux per reaction center (DI₀/RC) were also shown in the transgenic plants. Along with the decreased D1, CP47, and CP43 content, these results suggested photoinhibition under growth light conditions in transgenic plants. Decreased Deg1 caused inhibition of electron transfer on the PSII reducing side, leading to a decline in the number of Q_B-reducing centers and accumulation of Q_B-nonreducing centers. The Tm of the Q band shifted from 5.7 °C in the wild-type plant to 10.4 °C and 14.2 °C in the deg1-2 and deg1-4 plants, respectively, indicating an increase in the stability of S₂Q_A^¯ in transgenic plants. PSIIα in the transgenic plants largely reduced, while PSIIβ and PSIIγ increased with the decline in the Deg1 levels in transgenic plants suggesting PSIIα centers gradually converted into PSIIβ and PSIIγ centers in the transgenic plants. Besides, the connectivity of PSIIα and PSIIβ was downregulated in transgenic plants. Our results reveal that downregulation of Deg1 protein levels induced photoinhibition in transgenic plants, leading to loss of PSII activities on both the donor and acceptor sides in transgenic plants. These results give a new insight into the regulation role of Deg1 in PSII electron transport.

     

Characterization of the wave phenomenon of flash-induced chlorophyll fluorescence in Chlamydomonas reinhardtii

Flash-induced chlorophyll fluorescence relaxation is a powerful tool to monitor the reoxidation reactions of the reduced primary quinone acceptor, Q_A^? by Q_B and the plastoquinone (PQ) pool, as well as the charge recombination reactions between the donor and acceptor side components of Photosystem II (PSII). Under certain conditions, when the PQ pool is highly reduced (e.g. in microaerobic conditions), a wave phenomenon appears in the fluorescence relaxation kinetics, which reflects the transient reoxidation and re-reduction of Q_A^? by various electron transfer processes, which in cyanobacteria is mediated by NAD(P)H dehydrogenase (NDH-1). The wave phenomenon was also observed and assigned to the operation of type 2 NAD(P)H dehydrogenase (NDH-2) in the green alga Chlamydomonas reinhardtii under hydrogen-producing conditions, which required a long incubation of algae under sulphur deprivation (Krishna et al. J Exp Bot 70 (21):6321–6336, 2019). However, the conditions that induce the wave remained largely uncharacterized so far in microalgae. In this work, we investigated the wave phenomenon in Chlamydomonas reinhardtii under conditions that lead to a decrease of PSII activity by applying hydroxylamine treatment, which impacts the donor side of PSII in combination with a strongly reducing environment of the PQ pool (microaerobic conditions). A similar wave phenomenon could be induced by photoinhibitory conditions (illumination with strong light in the presence of the protein synthesis inhibitor lincomycin). These results indicate that the fluorescence wave phenomenon is activated in green algae when the PSII activity decreases relative to Photosystem I (PS I) activity and the PQ pool is strongly reduced. Therefore, the fluorescence wave could be used as a sensitive indicator of altered intersystem electron transfer processes, e.g. under stress conditions.

     

Excitonic connectivity between photosystem II units: what is it, and how to measure it?

In photosynthetic organisms, light energy is absorbed by a complex network of chromophores embedded in light-harvesting antenna complexes. In photosystem II (PSII), the excitation energy from the antenna is transferred very efficiently to an active reaction center (RC) (i.e., with oxidized primary quinone acceptor Q _A), where the photochemistry begins, leading to O₂ evolution, and reduction of plastoquinones. A very small part of the excitation energy is dissipated as fluorescence and heat. Measurements on chlorophyll (Chl) fluorescence and oxygen have shown that a nonlinear (hyperbolic) relationship exists between the fluorescence yield (Φ _F ) (or the oxygen emission yield, $ \Phi _{{{\text{O}}_{2} }} $ ) and the fraction of closed PSII RCs (i.e., with reduced Q _A). This nonlinearity is assumed to be related to the transfer of the excitation energy from a closed PSII RC to an open (active) PSII RC, a process called PSII excitonic connectivity by Joliot and Joliot (CR Acad Sci Paris 258: 4622–4625, 1964). Different theoretical approaches of the PSII excitonic connectivity, and experimental methods used to measure it, are discussed in this review. In addition, we present alternative explanations of the observed sigmoidicity of the fluorescence induction and oxygen evolution curves.     

Multiple sites of retardation of electron transfer in Photosystem II after hydrolysis of phosphatidylglycerol

Phosphatidylglycerol (PG), containing the unique fatty acid Δ3, trans-16:1-hexadecenoic acid, is a minor but ubiquitous lipid component of thylakoid membranes of chloroplasts and cyanobacteria. We investigated its role in electron transfers and structural organization of Photosystem II (PSII) by treating Arabidopsis thaliana thylakoids with phospholipase A₂ to decrease the PG content. Phospholipase A₂ treatment of thylakoids (a) inhibited electron transfer from the primary quinone acceptor Q_A to the secondary quinone acceptor Q_B, (b) retarded electron transfer from the manganese cluster to the redox-active tyrosine Z, (c) decreased the extent of flash-induced oxidation of tyrosine Z and dark-stable tyrosine D in parallel, and (d) inhibited PSII reaction centres such that electron flow to silicomolybdate in continuous light was inhibited. In addition, phospholipase A₂ treatment of thylakoids caused the partial dissociation of (a) PSII supercomplexes into PSII dimers that do not have the complete light-harvesting complex of PSII (LHCII); (b) PSII dimers into monomers; and (c) trimers of LHCII into monomers. Thus, removal of PG by phospholipase A₂ brings about profound structural changes in PSII, leading to inhibition/retardation of electron transfer on the donor side, in the reaction centre, and on the acceptor side. Our results broaden the simple view of the predominant effect being on the Q_B-binding site.     

EFFECT OF CHROMIUM(VI) ON PHOTOSYSTEM II ACTIVITY AND HETEROGENEITY OF SYNECHOCYSTIS SP. (CYANOPHYTA): STUDIED WITH IN VIVO CHLOROPHYLL FLUORESCENCE TESTS1

The inhibitory effect of Cr(VI) on the PSII of Synechocystis sp. was studied. Cr(VI) reduced O₂ evolution and inhibited the water‐splitting system in PSII. S‐states test and flash induction test showed that Cr(VI) exposure increased the proportion of inactivated PSII (PSII_X) and PSII_β reaction centers, which increased the fluxes of dissipated energy. JIP test and Q_A^? reoxidation test demonstrated that Cr(VI) treatment induces inhibition of electron transport from Q_A^? to Q_B/Q_B^? and accumulation of P₆₈₀⁺. More Q_A^? had to be oxidized through S₂(Q_AQ_B)^? charge recombination and oxidation by PQ9 molecules in PSII under Cr(VI) stress. These changes finally decreased the index of photosynthesis performance.     

Control of the maximal chlorophyll fluorescence yield by the Q<Subscript>B</Subscript> binding site

Differences in maximal yields of chlorophyll variable fluorescence (F_m) induced by single turnover (ST) and multiple turnover (MT) excitation are as great as 40%. Using mutants of Chlamydomonas reinhardtii we investigated potential mechanisms controlling F_m above and beyond the Q_A redox level. F_m was low when the Q_B binding site was occupied by PQ and high when the Q_B binding site was empty or occupied by a PSII herbicide. Furthermore, in mutants with impaired rates of plastoquinol reoxidation, F_m was reached rapidly during MT excitation. In PSII particles with no mobile PQ pool, F_m was virtually identical to that obtained in the presence of PSII herbicides. We have developed a model to account for the variations in maximal fluorescence yields based on the occupancy of the Q_B binding site. The model predicts that the variations in maximal fluorescence yields are caused by the capacity of secondary electron acceptors to reoxidize Q_A^–.     

PHOTOPHYSIOLOGICAL RESPONSES OF FRAGILARIOPSIS CYLINDRUS (BACILLARIOPHYCEAE) TO NITROGEN DEPLETION AT TWO TEMPERATURES1

The photosynthetic efficiency and photoprotective capacity of the sea‐ice diatom, Fragilariopsis cylindrus (Grunow) W. Krieg., grown in a matrix of nitrogen repletion and depletion at two different temperatures (?1°C and +6°C) was investigated. Temperature showed no significant effect on photosynthetic efficiency or photoprotection in F. cylindrus. Cultures under nitrogen depletion showed enhanced photoprotective capacity with an increase in nonphotochemical quenching (NPQ) when compared with nitrogen‐replete cultures. This phenomenon was achieved at no apparent cost to the photosynthetic efficiency of PSII (F_V/F_M). Nitrogen depletion yielded a partially reduced electron transport chain in which maximum fluorescence (F_M) could only be obtained by adding 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea (DCMU). reoxidation curves showed the presence of Q_B nonreducing PSII centers under nitrogen depletion. Fast induction curves (FICs) and electron transport rates (ETRs) revealed slowing of the electrons transferred from the primary (Q_A) to the secondary (Q_B) quinone electron acceptors of PSII. The data presented show that nitrogen depletion in F. cylindrus leads to the formation of Q_B nonreducing PSII centers within the photosystem. On a physiological level, the formation of Q_B nonreducing PSII centers in F. cylindrus provides the cell with protection against photoinhibition by facilitating the rapid induction of NPQ. This strategy provides an important ecological advantage, especially during the Antarctic spring, maintaining photosynthetic efficiency under high light and nutrient‐limiting conditions.     

A non-invasive assay of the plastoquinone pool redox state based on the OJIP-transient

The plastoquinone (PQ) pool of the photosynthetic electron transport chain becomes reduced under anaerobic conditions. Here, anaerobiosis was used as a tool to manipulate the PQ-pool redox state in darkness and to study the effects of the PQ-redox state on the Chl-a fluorescence (OJIP) kinetics in pea leaves (Pisum sativum L.). It is shown that the F_J (fluorescence intensity at 3 ms) is linearly related to the area above the OJ-phase (first 3 ms) representing the reduction of the acceptor side of photosystem II (PSII) and F_J is also linearly related to the area above the JI-phase (3–30 ms) that parallels the reduction of the PQ-pool. This means that F_J depends on the availability of oxidized PQ-molecules bound to the Q_B-site. The linear relationships between F_J and the two areas indicate that F_J is not sensitive to energy transfer between PSII-antennae (connectivity). It is further shown that a ∼94% reduced PQ-pool is in equilibrium with a ∼19% reduction of Q_A (primary quinone acceptor of PSII). The non-linear relationship between the initial fluorescence value (F_{20 μs}) and the area above the OJ-phase supports the idea that F_{20 μs} is sensitive to connectivity. This is reinforced by the observation that this non-linearity can be overcome by transforming the F_{20 μs}-values into [Q_A ⁻]-values. Based on the F_J-value of the OJIP-transient, a simple method for the quantification of the redox state of the PQ-pool is proposed. Szilvia Z. Tóth and Gert Schansker contributed equally to this study.     

DNA DAMAGE AND PHOTOSYNTHETIC PERFORMANCE IN THE ANTARCTIC TERRESTRIAL ALGA PRASIOLA CRISPA SSP. ANTARCTICA (CHLOROPHYTA) UNDER MANIPULATED UV-B RADIATION

The effect of reduced, natural ambient, and enhanced UV-B radiation (UVBR) on photosynthesis and DNA damage in the Antarctic terrestrial alga Prasiola crispa ssp. antarctica (Kützing) Knebel was investigated in two field experiments. Samples of P. crispa were collected underneath snow cover and exposed outside to reduced and natural UVBR in the austral spring. In a second experiment at the end of the austral summer, samples were exposed to ambient and enhanced UVBR. PSII efficiency, net photosynthetic rate (NP), dark respiration rate (DR), UV-absorbing pigments, and cyclobutyl pyrimidine dimer (CPD) formation were measured during the experiments. In October 1998, a spring midday maximum of 2.0 W·m ^{− 2} of UVBR did not significantly affect effective quantum yield (ΔF/F_m′), and a reduction in the ratio of variable to maximal fluorescence (F_v/F_m) in the late afternoon was transient. Exposure to natural ambient UVBR in October increased CPD values significantly. Midday maxima of UVBR during the experiments in October and January were comparable, but Setlow-DNA-weighted UVBR was more than 50% lower in January than in October. In January, 0.5 W·m ^{− 2} additional UVBR during 10 h did not have a negative effect on ΔF/F_m′. The reduction in F_v/F_m was not significant. NP and DR were not affected by supplementation of UVBR. Although photosynthetic activity remained largely unaffected by UVBR treatment, DNA damage was shown to be a sensitive parameter to monitor UVBR effects. Supplementation of additional UVBR did significantly enhance the amounts of CPD in exposed samples and repair took place overnight. It is concluded that PSII and whole-chain photosynthesis of P. crispa is well adapted to ambient and enhanced levels of UVBR but that CPD formation is more sensitive to UVBR than to photosynthesis.     

EFFECTS OF POTASSIUM ON THE PHOTOSYNTHETIC RECOVERY OF THE TERRESTRIAL CYANOBACTERIUM,NOSTOC FLAGELLIFORME (CYANOPHYCEAE) DURING REHYDRATION1

Effects of potassium on the photosynthetic recovery of Nostoc flagelliforme (Berk. & Curtis) Bornet & Flahault were investigated to determine its exact role during rehydration. Potassium enhanced recovery of the ability to reduce the primary quinone‐type acceptor (Q_A) and plastoquinone (PQ) pool and the area over the fluorescence rise curve was increased by 127%. The proportions of closed PSII reaction centers at phases J and I and the net rate of closure of PSII reaction centers were decreased by, respectively, 19%, 8%, and 23% with the addition of potassium, due to changes in the ability of PSII for multiple turnovers needed to reduce the PQ pool. Potassium significantly enhanced the probability of electron transfer beyond Q_A and the recovery of electron transport flux per PSII reaction center. Electron transport from water to methyl viologen for samples rehydrated in K⁺‐free BG₁₁ medium was 54% of those with the addition of potassium. However, electron flow from water to p‐benzoquinone and from reduced 2,6‐dichlorophenol‐indophenol to methyl viologen showed little change with the addition of potassium. The fast phase and slow phase of millisecond delayed light emission and the ATP content for samples rehydrated in K⁺‐free BG₁₁ medium were, respectively, 71.6%, 50.7%, and 77.1% of those with the addition of potassium. These suggested that potassium affected electron transfer from PQ to plastocyanin through the cytochrome b₆f complex and the proton motive force across the thylakoid membranes, probably reflecting its role in charge balance during H⁺ transport by the cytochrome b₆f complex.     

Properties of inactive Photosystem II centers

A fraction (usually in the range of 10–25%) of PS II centers is unable to transfer electrons from the primary quinone acceptor Q_A to the secondary acceptor Q_B. These centers are inactive with respect to O₂ evolution since their reopening after photochemical charge separation to the S₂O_A ^- state involves predominantly a back reaction to S₁Q_A in the few seconds time range (slower phases are also occurring). Several properties of these centers are analyzed by fluorescence and absorption change experiments. The initial rise phase F_o-F_pl of fluorescence induction under weak illumination reflects both the closure of inactive centers and the modulation of the fluorescence yield by the S-states of the oxygen-evolving system: We estimate typical relative amplitudes of these contributions as, respectively, 65 and 35% of the F_o-F_pl amplitude. The half-rise time of this phase is significantly shorter than for the fluorescence induction in the presence of DCMU (in which all centers are involved). This finding is shown to be consistent with inactive centers sharing the same light-harvesting antenna as normal centers, a view which is also supported by comparing the dependence of the fluorescence yield on the amount of closed active or inactive centers estimated through absorption changes. It is argued that the exponential kinetics of the F_o-F_pl phase does not indicate absence of excitation energy transfer between the antennas of inactive and active centers. We show that the acceptor dichlorobenzoquinone does not restore electron transfer in inactive centers, in disagreement with previous suggestions. We confirm, however, the enhancement of steady-state electron flow caused by this quinone and suggest that it acts by relieving a blocking step involved in the reoxidation of a fraction of the plastoquinone pool. Part of the discrepancies between the present results and those from previous literature may arise from the confusion of inactive centers characterized on a single turnover basis and PS II centers that become blocked under steady-state conditions because of deficient reoxidation of their secondary acceptors.Abbreviations DCBQ 2,6-dichloro-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DMQ 2,5-dimethyl-p-benzoquinone - PS photosystem     

Reduction of QA in the dark: Another cause of fluorescence Fo increases by high temperatures in higher plants

Increases in the chlorophyll fluorescence F_o (dark level fluorescence) during heat treatments were studied in various higher plants. Besides the dissociation of light-harvesting chlorophyll a/b protein complexes from the reaction center complex of PS II and inactivation of PS II, dark reduction of Q_A via plastoquinone (PQ) seemed to be related to the F_o increase at high temperatures. In potato leaves or green tobacco cultured cells, a part of the F_o increase was quenched by light, reflecting light-induced oxidation of Q_A ^- which had been reduced in the dark at high temperatures. Appearance of the F_o increase due to Q_A reduction depended on the plant species, and the mechanisms for this are proposed. The reductants seemed to be already present and formed by very brief illumination of the leaves at high temperatures. A ndhB-less mutant of tobacco showed that complex I type NAD(P)H dehydrogenase is not involved in the heat-induced reduction of Q_A. Quite strong inhibition of the Q_A reduction by diphenyleneiodonium suggests that a flavoenzyme is one of the electron mediator to PQ from the reductant in the stroma. Reversibility of the heat-induced Q_A reduction suggests that an enzyme(s) involved is activated at high temperatures and mostly returns to an inactive form at room temperature (25 °C).This revised version was published online in October 2005 with corrections to the Cover Date.