Mol Chl Research Articles

Salt and heat stress enhances hydrogen production in cyanobacteria.

Cyanobacteria are among the most suitable organisms for the capture of excessive amounts of CO2 and can be grown in extreme environments. In our research we use the single-celled freshwater cyanobacteria Synechococcus elongatus PCC7942 PAMCOD strain and Synechocystis sp. PCC6714 for the production of carbohydrates and hydrogen. PAMCOD strain and Synechocystis sp. PCC6714 synthesize sucrose when exposed to salinity stress, as their main compatible osmolyte. We examined the cell proliferation rate and the sucrose accumulation in those two different strains of cyanobacteria under salt (0.4M NaCl) and heat stress (35 0C) conditions. The intracellular sucrose (mol sucrose content per Chl a) was found to increase by 50% and 108% in PAMCOD strain and Synechocystis sp. PCC6714 cells, respectively. As previously reported, PAMCOD strain has the ability to produce hydrogen through the process of dark anaerobic fermentation (Vayenos D, Romanos GE, Papageorgiou GC, Stamatakis K (2020) Photosynth Res 146, 235-245). In the present study, we demonstrate that Synechocystis sp. PCC6714 has also this ability. We further examined the optimal conditions during the dark fermentation of PAMCOD and Synechocystis sp. PCC6714 regarding H2 formation, increasing the PAMCOD H2 productivity from 2 nmol H2 h- 1 mol Chl a- 1 to 23 nmol H2 h- 1 mol Chl a- 1. Moreover, after the dark fermentation, the cells demonstrated proliferation in both double BG-11 and BG-11 medium enriched in NaNO3, thus showing the sustainability of the procedure.

Hydrogen production through oxygenic photosynthesis using the cyanobacterium Synechocystis sp. PCC 6803 in a bio-photoelectrolysis cell (BPE) system

Microbial electrolysis cells (MECs) represent an emerging technology that uses heterotrophic microbes to convert organic substrates into fuel products, such as hydrogen gas (H2). The recent development of biophotovoltaic cells (BPVs), which use autotrophic microbes to produce electricity with only light as a substrate, raises the possibility of exploiting similar systems to harness photosynthesis to drive the production of H2. In the current study we explore the capacity of the cyanobacterium Synechocystis sp. PCC 6803 to generate electrons by oxygenic photosynthesis and facilitate H2 production in a two-chamber bio-photoelectrolysis cell (BPE) system using the electron mediator potassium ferricyanide ([Fe(CN)6]3−). The performance of a wild-type and mutant strain lacking all three respiratory terminal oxidase activities (rto) was compared under low or high salt conditions. The rto mutant showed a decrease in maximum photosynthetic rates under low salt (60% lower Pmax than wild-type) but significantly increased rates under high salt, comparable to wild-type levels. Remarkably, rto demonstrated a 3-fold increase in (Fe[CN]6)3− reduction rates in the light under both low and high salt compared to the wild-type. Yields of H2 and efficiency parameters were similar between wild-type and rto, and highest under high salt conditions, resulting in a maximum rate of H2 production of 2.23 ± 0.22 ml H2 l−1 h−1 (0.68 ± 0.11 mmol H2 [mol Chl]−1 s−1). H2 production rates were dependent on the application of a bias-potential, but all voltages used were significantly less than that required for water electrolysis. These results clearly show that production of H2 using cyanobacteria is feasible without the need to inhibit photosynthetic O2 evolution. Optimising the balance between the rates of microbial-facilitated mediator reduction with H2 production may lead to long-term sustainable H2 yields.

Sun-shade patterns of leaf carotenoid composition in 86 species of neotropical forest plants.

A survey of photosynthetic pigments, including 86 species from 64 families, was conducted for leaves of neotropical vascular plants to study sun-shade patterns in carotenoid biosynthesis and occurrence of α-carotene (α-Car) and lutein epoxide (Lx). Under low light, leaves invested less in structural components and more in light harvesting, as manifested by low leaf dry mass per area (LMA) and enhanced mass-based accumulation of chlorophyll (Chl) and carotenoids, especially lutein and neoxanthin. Under high irradiance, LMA was greater and β-carotene (β-Car) and violaxanthin-cycle pool increased on a leaf area or Chl basis. The majority of plants contained α-Car in leaves, but the α- to β-Car ratio was always low in the sun, suggesting preference for β-Car in strong light. Shade and sun leaves had similar β,ε-carotenoid contents per unit Chl, whereas sun leaves had more β,β-carotenoids than shade leaves. Accumulation of Lx in leaves was found to be widely distributed among taxa: >5 mmol mol Chl-1 in 20% of all species examined and >10 mmol mol Chl-1 in 10% of woody species. In Virola elongata (Benth.) Warb, having substantial Lx in both leaf types, the Lx cycle was operating on a daily basis although Lx restoration in the dark was delayed compared with violaxanthin restoration.

Nitrogen fixation and growth rates of Trichodesmium IMS-101 as a function of light intensity

MEPS Marine Ecology Progress Series Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections MEPS 359:25-36 (2008) - DOI: https://doi.org/10.3354/meps07241 Nitrogen fixation and growth rates of Trichodesmium IMS-101 as a function of light intensity E. Breitbarth1,2,*, J. Wohlers1, J. Kläs1, J. LaRoche1, I. Peeken1 1Leibniz-Institute of Marine Sciences, IFM-GEOMAR, Düsternbrooker Weg 20, 24105 Kiel, Germany 2Present address: Department of Chemistry, Analytical and Marine Chemistry, University of Gothenburg, Kemivägen 10, 412 96 Göteborg, Sweden *Email: eike@chem.gu.se ABSTRACT: The diazotrophic cyanobacterium Trichodesmium is a significant contributor to marine nitrogen and carbon cycles and has been incorporated in biogeochemical ocean circulation models. To date, parameterization of light as a controlling factor for nitrogen fixation has been based on field observations, where factors other than light also affect Trichodesmium physiology. Here we present data on light-dependent (15 to 1100 µmol quanta m–2 s–1) diazotrophic growth from controlled laboratory experiments and their implications for modeling approaches. We supply a simple empirical model to describe nitrogen fixation by Trichodesmium in batch cultures. Diazotrophic growth of axenic Trichodesmium IMS-101 was light saturated at 180 µmol quanta m–2 s–1 and did not vary significantly at higher photon irradiances up to 1100 µmol quanta m–2 s–1 (μcarbon based ≈ 0.26 d–1). Chlorophyll a (chl a) normalized N2 fixation rates were significantly affected by light intensity during mid-exponential growth (0.74 to 4.45 mol N fixed mol chl a–1 h–1) over the range of photon irradiances tested. In contrast, nitrogen fixation rates normalized to the cellular carbon content were relatively unaffected by light intensity (0.42 to 0.59, averaging 0.5 mmol N mol particulate organic carbon [POC]–1 h–1). Trichodesmium carbon biomass can be used to estimate the nitrogen input by this diazotroph into the ocean; the maximum input rate is 350 nmol N fixed l–1 h–1. KEY WORDS: Trichodesmium · Light · Nitrogen fixation · Marine nitrogen cycle · Marine carbon cycle · Marine cyanobacteria · Diazotrophic growth Full text in pdf format Supplementary appendix PreviousNextCite this article as: Breitbarth E, Wohlers J, Kläs J, LaRoche J, Peeken I (2008) Nitrogen fixation and growth rates of Trichodesmium IMS-101 as a function of light intensity. Mar Ecol Prog Ser 359:25-36. https://doi.org/10.3354/meps07241Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in MEPS Vol. 359. Online publication date: May 05, 2008 Print ISSN: 0171-8630; Online ISSN: 1616-1599 Copyright © 2008 Inter-Research.

Impact of climate forcing on ecosystem processes in the North Pacific Subtropical Gyre

Measurements at the Hawaii Ocean Time‐series (HOT) Station ALOHA (22°45′N, 158°W) have revealed a significant, approximately 50% increase in euphotic zone depth‐integrated rates of primary production (PP; mol C fixed m−2 d−1) based on in situ 14C experiments. The character of the nearly two‐decade increasing trend in PP was punctuated by several abrupt episodes that coincided with changes in the El Niño/Southern Oscillation (ENSO), and the Pacific Decadal Oscillation (PDO) climate indices, or both. In contrast to the observed increase in rates of PP, the PP per unit chlorophyll a (mol C fixed mol chl a−1 d−1), a measure of the biomass‐normalized production, was relatively constant, whereas PP per unit solar radiation (mol C fixed mol quanta−1), a measure of the efficiency of light utilization, varied in synchrony with the temporal trend in PP. Coincident with variations in PP, the HOT program core data sets also revealed changes in mixed‐layer depth, upper ocean stratification, inorganic nutrients, phototrophic microbial abundances and pigment inventories. These time‐series data suggest that the ENSO/PDO may control upper ocean stratification and vertical nutrient delivery into the euphotic zone at Sta. ALOHA, thereby influencing the composition of the plankton assemblage and altering rates of PP and particulate matter export.

Effects of Cold-Hardening on Chilling-Induced Photoinhibition of Photosynthesis and on Xanthophyll Cycle Pigments in Sweet Pepper

Two cultivars of Capsicum annuum L. were acclimated for 5 d at sub-optimal temperature (14 °C) and irradiance of 250 µmol m−2 s−1. This cold-hardening resulted in some reduction in the extent of photoinhibition during an 8 h exposure to high irradiance at 4 °C. Obvious differences were observed between non-hardened leaves (NHL) and cold-hardened leaves (CHL) in the recovery under low irradiance at room temperature. The CHL of both cultivars recovered faster than NHL, especially during the initial fast phase of recovery. Compared with NHL, the total content of carotenoids (Cars), based on chlorophyll, Chl (a+b), and the proportions of xanthophyll cycle pigments referred to total Cars increased in CHL, mainly due to an increase of violaxanthin (V) + antheraxanthin (A) + zeaxanthin (Z) content per mol Chl (a+b). Faster development and a higher non-photochemical quenching (NPQ) of Chl fluorescence, related to a stronger deepoxidation of the larger xanthophyll cycle pool in NHL, could act as a major defence mechanism to reduce the formation of reactive oxygen species during severe chilling. This is suggested by higher content of Z or Z+A in photoinhibition as well as by its rapid decline during the initial fast phase of recovery. In contrast to the chilling-sensitive cv. 0004, the chilling-tolerant cv. 1141 did more easily acclimate its photosynthetic apparatus to low temperatures.

Cell turgor: A critical factor for the proliferation of cyanobacteria at unfavorable salinity.

We employed chlorophyll a fluorometry in order to measure the evolution of turgor threshold (intracellular osmolality) during the adaptation of two genetic transformants of the freshwater cyanobacterium Synechococcus sp. PCC7942 to unfavorable external salinity: PAMCOD cells which oxidize imported choline and accumulate approx. 0.06-0.08 M glycine betaine; and PAM cells which do not oxidize choline [Deshnium et al. (1995a) Plant Mol Biol 29: 897-909]. Turgor thresholds increased linearly (a) with the NaCl concentration in the culture, and (b) with the molar sucrose/chlorophyll a ratio in the cell. PAMCOD cells could proliferate in culture medium containing 0.4 M NaCl (external osmolality, 0.815 Osm kg(-1)), after a lag period, during which intracellular sucrose rose to 10 mol (mol Chl a)(-1), or more, and turgor threshold (cytoplasmic osmolality) exceeded 1 Osm kg(-1). At comparative conditions, PAM cells accumulated approx. half as much sucrose, and attained approx. half as high turgor thresholds as the PAMCOD cells, but they did not proliferate. These results indicate that glycine betaine improved the salinity tolerance of the PAMCOD cells synergistically, by means of two effects that implicate sucrose, the main organic osmolyte of Synechocccus: enhancement of sucrose biosynthesis, and/or alleviation of sucrose toxicity.

Dunaliella salina (Chlorophyta) with small chlorophyll antenna sizes exhibit higher photosynthetic productivities and photon use efficiencies than normally pigmented cells

The photon use efficiencies and maximal rates of photosynthesis in Dunaliella salina (Chlorophyta) cultures acclimated to different light intensities were investigated. Batch cultures were grown to the mid-exponential phase under continuous low-light (LL: 100 μmol photon m-2 s-1) or high-light (HL: 2000 μmol photon m-2 s-1) conditions. Under LL, cells were normally pigmented (deep green) containing ∼500 chlorophyll (Chl) molecules per photosystem II (PSII) unit and ∼250 Chl molecules per photosystem I (PSI). HL-grown cells were yellow-green, contained only 60 Chl per PSII and 100 Chl per PSI and showed signs of chronic photoinhibition, i.e., accumulation of photodamaged PSII reaction centers in the chloroplast thylakoids. In LL-grown cells, photosynthesis saturated at ∼200 μmol photon m-2 s-1 with a rate (Pmax) of ∼100 mmol O2 (mol Chl)-1 s-1. In HL-grown cells, photosynthesis saturated at much higher light intensities, i.e. ∼2500 μmol photon m-2 s-1, and exhibited a three-fold higher Pmax (∼300 mmol O2 (mol Chl)-1 s-1) than the normally pigmented LL-grown cells. Recovery of the HL-grown cells from photoinhibition, occurring prior to a light-harvesting Chl antenna size increase, enhanced Pmax to ∼675 mmol O2 (mol Chl)-1 s-1. Extrapolation of these results to outdoor mass culture conditions suggested that algal strains with small Chl antenna size could exhibit 2–3 times higher productivities than currently achieved with normally pigmented cells.

Changes in the quantities of violaxanthin de-epoxidase, xanthophylls and ascorbate in spinach upon shift from low to high light

Zeaxanthin, a carotenoid in the xanthophyll cycle, has been suggested to play a role in the protection against photodestruction. We have studied the importance of the parameters involved in zeaxanthin formation by comparing spinach plants grown in low light (100 to 250 μmol m-2 s-1) to plants transferred to high light (950 μmol m-2 s-1). Different parameters were followed for a total of 11 days. Our experiments show that violaxanthin de-epoxidase decreased between 15 and 30%, the quantity of xanthophyll cycle pigments doubled to 100 mmol (mol Chl)-1, corresponding to 27 μmol m-2, and the rate of violaxanthin to zeaxanthin conversion was doubled. Lutein and neoxanthin increased from 50 to 71 μmol m-2 and from 16 to 23 μmol m-2, respectively. On a leaf area basis, chlorophyll and β-carotene levels first decreased and then after 4 days increased. The chlorophyll a/b ratio was unchanged. The quantity of ascorbate was doubled to 2 mmol m-2, corresponding to an estimated increase in the chloroplasts from 25 to 50 mM. In view of our data, we propose that the increase in xanthophyll cycle pigments and ascorbate only partly explain the increased rate of conversion of violaxanthin to zeaxanthin, but the most probable explanation of the faster conversion is an increased accessibility of violaxanthin in the membrane.

Carbon Fixation Profiles Do Reflect Light Absorption Profiles in Leaves

Bifacial leaves contain an array of chloroplasts which are aligned along cell walls adjacent to intercellular airspaces. Cells beneath the adaxial (upper) surface are generally cylindrical in shape, forming palisade tissue and contain chloroplasts that have characteristics associated with acclimation to high irradiance. Conversely, cells near the abaxial (lower) surface are irregular in shape, forming spongy tissue and contain chloroplasts that have characteristics associated with acclimation to low irradiance. This results in a gradient of declining photosynthetic capacity per unit chlorophyll with increasing depth into the leaf. Space irradiance declines dramatically through the leaf. The profile of light absorption through the leaf has yet to be measured, but should approximate the product of space irradiance and pigment profiles. Since pigment content is highest in the middle of the leaf, light absorption peaks about one quarter of the way through the leaf. The response of leaf photosynthetic rate to irradiance reflects the interaction between the profiles of light absorption and chloroplast characteristics. Quantum yield (mol CO2 mol-1 quanta absorbed) varies for different layers in the leaf because the profile of Rubisco content per unit chlorophyll is not as steep as the light absorption profile. This skews the profile of carbon fixation away from the light absorption profile, favouring greater fixation in deeper layers. Nishio, Sun and Vogelmann (1993, Plant Cell 5, 953-961) measured profiles of 14C fixation across spinach leaves and concluded that carbon fixation was disconnected from the light gradient. Reanalysis of their data shows that carbon fixation is remarkably consistent with light absorption obeying the Beer- Lambert law when allowance is made for chlorophyll and Rubisco profiles through the leaf. This interpretation is supported by chlorophyll fluorescence properties of adaxial and abaxial surfaces. An apparent extinction coefficient of about 1500 m2 (mol Chl)-1 could be derived from leaf data which compares with 2230 m2 (mol Chl)-1 for pigment-protein complexes in solution. A possible test for this analysis would be to measure the 14C fixation profile of leaves illuminated on their abaxial surface.

Temperature dependence of violaxanthin de-epoxidation and non-photochemical fluorescence quenching in intact leaves of Gossypium hirsutum L. and Malva parviflora L.

The temperature dependence of the rate of de-epoxidation of violaxanthin to zeaxanthin was determined in leaves of chilling-sensitive Gossypium hirsutum L. (cotton) and chilling-resistant Malva parviflora L. by measurements of the increase in absorbance at 505 nm (ΔA 505) and in the contents of antheraxanthin and zeaxanthin that occur upon exposure of predarkened leaves to excessive light. A linear relationship between ΔA 505 and the decrease in the epoxidation state of the xanthophyll-cycle pigment pool was obtained over the range 10-40° C. The maximal rate of de-epoxidation was strongly temperature dependent; Q10 measured around the temperature at which the leaf had developed was 2.1-2.3 in both species. In field-grown Malva the rate of de-epoxidation at any given measurement temperature was two to three times higher in leaves developed at a relatively low temperature in the early spring than in those developed in summer. Q10 measured around 15° C was in the range 2.2-2.6 in both kinds of Malva leaves, whereas it was as high as 4.6 in cotton leaves developed at a daytime temperature of 30° C. Whereas the maximum (initial) rate of de-epoxidation showed a strong decrease with decreased temperature the degree of de-epoxidation reached in cotton leaves after a 1-2 · h exposure to a constant photon flux density increased with decreased temperature as the rate of photosynthesis decrease. The zeaxanthin content rose from 2 mmol · (mol chlorophyll)(-1) at 30° C to 61 mmol · (mol Chl)(-1) at 10° C, corresponding to a de-epoxidation of 70% of the violaxanthin pool at 10° C. The degree of de-epoxidation at each temperature was clearly related to the amount of excessive light present at that temperature. The relationship between non-photochemical quenching of chlorophyll fluorescence and zeaxanthin formation at different temperatures was determined for both untreated control leaves and for leaves in which zeaxanthin formation was prevented by dithiothreitol treatment. The rate of development of that portion of non-photochemical quenching which was inhibited by dithiothreitol decreased with decreasing temperature and was linearly related to the rate of zeaxanthin formation over a wide temperature range. In contrast, the rate of development of the dithiothreitol-resistant portion of non-photochemical quenching was remarkably little affected by temperature. Evidently, the kinetics of the development of non-photochemical quenching upon exposure of leaves to excessive light is therefore in large part determined by the rate of zeaxanthin formation. For reasons that remain to be determined the relaxation of dithiothreitolsensitive quenching that is normally observed upon darkening of illuminated leaves was strongly inhibited at low temperatures.

Dynamics of Photosystem II heterogeneity during photoinhibition: depletion of PS II β from non-appressed thylakoids during strong-irradiance exposure of Chlamydomonas reinhardtii

Thylakoid membranes of Chlamydomonas reinhardtii were fractionated into appressed membranes and non-appressed vesicles. The latter had a Chl a/b ratio of 3.2 and contained 1.12 mmol PS II (mol Chl a+b)−1. The PS II centers in the non-appressed regions displayed the kinetic characteristics of PS IIβ centers. They were able to generate a stable charge-separation to QA, but they were unable to transfer electrons from Q−A to QB (QB-nonreducing centers). After a strong-irradiance treatment (SIT) of cells by exposure to 2000 μmol · m−2 · s−1 for 15 min, a portion of the PS IIα centers in the appressed membranes was damaged due to photoinhibition. PS IIβ centers were resistant to photoinhibitory damage; however, SIT caused a lowering of the PS II content in the non-appressed fraction to 0.60 mmol PS II (mol Chl a+b)−1. Concomitantly, there was a conversion of QB-nonreducing centers to a form able to reduce the plastoquinone pool (QB-reducing centers). Low-temperature during SIT inhibited both the depletion of PS IIβ from the non-appressed membrane region and the activation of QB-nonreducing centers. These results are consistent with a ‘PS II repair cycle’ which translocates PS II units from the stroma-exposed membranes into the appressed thylakoids during activation of PS IIβ from a QB-nonreducing to a QB-reducing form. Since the overall distribution of Chl-protein complexes between the stroma-exposed and appressed membrane regions was unaffected by SIT, it is suggested that damaged PS II units from the appressed membrane region are exchanged with PS IIβ from the stroma-exposed membranes.

The Stoichiometries of Supramolecular Complexes in Thylakoid Membranes From Spinach Chloroplasts

The concentrations of photosystem II (PS II), photosystem I (PS I) and cytochrome b/f complexes on a chlorophyll basis have been determined for chloroplasts of spinach grown under three irradiances in a glasshouse. Assaying PS II by atrazine binding gave concentrations that exceeded the estimates from flash-induced O2 or H+ yield by a factor of 1.21 � 0.04 (23). Since part of this factor (1.14) is expected to arise from missed turnovers in excited reaction centres, it is concluded that both methods are valid to determine [PS II] in gently isolated chloroplasts. The agreement between the methods also suggests that atrazine does not bind to additional sites of quenchers such as 'Q400', contrary to the suggestion of R. J. Dennenberg and P. A. Jursinic [Biochim. Biophys. Acta 808 (1985), 192-200]. Whilst [PS I] was independent of growth irradiances, [PS II] and [cyt f ] increased with irradiance, as did the latent ATPase activity, a measure of the concentration of coupling factor 1. If [PS I] is taken as constant at 1.65 mmol (mol Chl)-1, the mean stoichiometries of PS II : cyt b/f complex : PS I obtained at the minimum and maximum irradiances were 1.2 : 0.7 : 1 and 1.6 : 1.0 : 1 respectively, PS II being determined by atrazine binding.

Adsorption of Amines to Thylakoid Surfaces and Estimations of ΔpH

The adsorption of the fluorescent, weak amine ΔpH indicators 9-aminoacridine and N-(1-naphthyl)ethylenediamine to thylakoids of pea chloroplasts in the dark was measured as a function of amine concentration, at several pH's between 8 and 5, and with varied MgCl2 concentration. It was concluded that it was the protonated form of the amine that was adsorbed. Maximum amounts adsorbed, saturation occurring at about 1 mM amine concentration in the presence of 5 mM KCl and 10 mM MgCl2, were up to 1 mol (mol Chl)-1. This was calculated as exceeding the number of available surface negative charges (0.26 for a charge density of 0.025 C m-2). An apparent binding constant of 400-500 μM (concentration for half saturation) was noted for both amines under the above conditions. A model was developed that enabled the estimation of the relative amounts of amine bound to the inside and outside thylakoid surfaces in the steady state, in the light. By this means corrections to the apparent ΔpH calculated from fluorescence quenching of the amines could be established. These corrections amounted to about 1 pH unit for the diamine and more for 9-aminoacridine. The predictions of the model were in good agreement with experimental relations between estimated, light-induced ΔpH values and amine concentration. The implications of a lower ΔpH for earlier estimations of proton motive force in relation to photophosphorylation are briefly considered.

Proton Translocations in Isolated Spinach Chloroplasts after Single-turnover Actinic Flashes

Proton movements were monitored with cresol red and neutral red. Absorbance changes of these dyes following actinic flashes were measured both for the steady state and for sequences of 6-9 flashes given to separate dark-adapted suspensions of chloroplasts. The cresol red changes corresponded to a steady-state proton uptake (�H*+ss) of 0.0015-0.0017 mol H+/mol Chl per flash (ferricyanide as electron acceptor) or 0.0031 mol H+/mol Chl per flash (methyl viologen). Assuming 600 Chl per electron transport chain, these uptakes corresponded to H+/e- = 1 or 2 respectively. In flash sequences with ferricyanide, a minimum of about 0.6 �H*+ss was noted after flash No. 3. Controls, and considerations of the partitioning of unprotonated neutral red into the thylakoid membrane phase, strongly indicated that neutral red was responding to an acidification of the intrathylakoid aqueous phase. The steady-state signal was interpreted as 2 H+ deposited per electron transport chain per flash (H+/e- = 2), one proton from the oxidation of water and one from the oxidation of plastoquinone. Patterns of proton deposition following sequences of flashes and their change upon adding dibromothymoquinone suggested that (a) protons are produced one at a time in the advance of the 'S-states' (Z*n+ � Z(n+1)+ ); and (b) the precursor to the plastoquinone pool, B, is a special plastoquinone, requiring 1 e- and 1 H+ for its initial reduction but passing on to the plastoquinone pool 2 e- upon the next flash. The proton deposition on flash No. 1 corresponded to a small proportion of reduced B in dark-adapted chloroplasts, as previously postulated.