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The final chapter looks at how the “light” reactions in photosynthesis were elucidated in the 1950s. This pursuit culminated in the two-photoreaction, two-pigment system model, which still dominates the field. Three strands of research are identified that led to this model: (1) the investigation of how reducing power (today: nicotinamide adenine dinucleotide phosphate (NADP)) was synthesised; (2) the discovery of photophosphorylation, that is, the production of adenosine triphosphate (ATP), in plant cells; (3) the search for a photosynthetic electron transport chain. The development of new spectroscopic methods was highly important in this context, since, for the very first time, it became possible to nail down the changing redox states of individual molecules. It was also in this period that photosynthesis researchers first tried to localise the partial processes in the cellular environment. And they experimented with various forms of graphs to visualise the mechanism. In contrast to the study of the carbon reduction process, analysed in Chap. 6, several research teams independently worked on this question, and almost simultaneously came to very similar conclusions; major players including Louis N. M. Duysens, Bessel Kok, Robin Hill and Horst T. Witt. The convergence of their results, although they had very different starting points and methodologies, was enormously persuasive. The model subsequently became known as the “Z-scheme” (after a standard form of representation), and this is how it features in textbooks of the field to this day.

In this chapter, we describe the mechanisms of acido-tolerance in an acidophilic- and thermophilic red alga, Cyanidium caldarium. In spite of the extremely acidic environments it inhabits, the intracellular pH of Cyanidium cells is kept neutral by pumping out the protons previously leaked into the cells according to the steep pH gradient. The H+ pump is driven by the plasma membrane ATPase, utilizing intracellular ATP produced by both oxidative phosphorylation and cyclic photophosphorylation via photosystem I. We also describe the characteristics and function of the two photosystems, Photosystem I (PSI) and II (PSII), in Cyanidium caldarium in comparison with those of cyanobacteria, other eukaryotic algae, and higher plants, based on the crystal structures of the two complexes reported so far.

In this session on “Life without starlight” it may be useful to describe photosynthetic organisms with their light-induced transformation, conversion and conservation of energy as “life with starlight”. To what extent could photophosphorylation, this fundamental process of energy transfer in photosynthesis, have originated and evolved in discrete molecular steps in the absence of light? An answer may be sought in the first and so far only known alternative biological photophosphorylation system, where the comparatively “primitive” energy donor inorganic pyrophosphate (PPi) is produced (H. Baltscheffsky et al., 1966) instead of the more “complex” adenosine triphosphate (ATP), the well known central energy donor and “energy currency” of all now living cells. The gene for PPi synthase, which is the enzyme involved in the photosynthetic formation of PPi in chromatophores from the photosynthetic bacterium Rhodospirillum rubrum, has been cloned (M. Baltscheffsky et al., 1998) and the primary structure and some other properties of this first bacterial representative of the membrane-bound family of proton-pumping inorganic pyrophosphatases (H+-PPases) has been deduced (M. Baltscheffsky et al., 1999). In the loop between the fifth and sixth putative transmembrane segments, in what appears to be a pyrophosphate binding region and the active site for the phosphorylation reaction, three different “primitive” tetrapeptidyl motifs have been located in two nonapeptidyl sequences. Their high content of the four “very early” proteinaceous amino acids glycine, alanine, valine and aspartic acid (G, A, V and D, respectively) and their very regular arrangement of amino acids with charged side groups, would seem to be of particular evolutionary significance. Our first tentative model of the active site has resulted in a structure, which may be useful for attempts to extrapolate back to the hither toelusive molecular origin and evolution of the metabolism of phosphate compounds, specially those, which are directly involved in bioenergy conversion.

The CO2-content of the atmosphere is steadily increasing (1,2). It is generally assumed that the CO2-content of air will increase within the next 50 years from now 350 ppm to 700 ppm. In context with this expected atmospheric change it is of interest to know what impact the CO2-increase will have on growth of higher plants. With Nicotiana tabacum and the Chinese “Tung Oil Tree” Aleurites montana we were able to show that under the influence of the increased CO2-content 30–40% more biomass is formed (3,4,5). Differences in dependence on the CO2-content are observed for the chlorophyll and sugar content on the one hand but also with respect to the content of the biomass forming enzyme ribulose 1.5-bisphosphate carboxylase and the energy equivalent-forming coupling factor of photophosphorylation CF1. Molecular changes are observed in the composition of peptides of photosystem II and photosystem I. Thus, the light-harvesting complex of photosystem II (3,4) is reduced and the same is true for photosystem I (5). The oxygen-evolving-complex (OEC)-peptides are reduced in plants cultivated under increased CO2 (3,4). In addition the electron transport components (plastoquinone, cytochrome f, plastocyanin, ferredoxin, ferredoxin-NADP-reductase) are reduced under these conditions as shown by rocket-immune-electrophoresis (6).

Heat treatment (HT) of leaves, isolated chloroplasts or intact plants, causes inhibition of electron transport activities of both PSII and PSI (1). The order of thermal sensitivity of various photosynthetic light reactions is oxygen evolution, photochemical activity of PSII, Photophosphorylation, Cyt b6/f complex and photochemical activity of PSI (2). In vitro studies with isolated thylakoids suggest that loss of extrinsic protein namely 33 KDa Mn-stabilizing protein at the donor side of PSI1 and subsequent loss of Mn are the primary causes of inhibition of electron transport by heat stress (3). However, unlike the donor side, very few reports exist on the heat induced changes in the PSII acceptor side. Further, Michalski and Wettern,(4) observed that incubation of algal cells at 41.5 °C for 90 min resulted in membrane bound break down products of Dl-Polypeptide of 27 and 25 KDa. They suggest that heat shock might have led to these intermediates as a result of a heat-dependent inactivation of a protesase that degrades damaged Dl. They also reported that, D1 igestion during heat shock incubation in darkness resulted in an additional membrane bound intermediate of 15 KDa. Heat shock incubation in the presence of light did not induce any degradation products of D2 while treatment in dark led to the formation of two degradation products of 26 KDa. These results prompted us to study the degradation pattern of D1 and D2 upon in vivo HT in higher plants and also QA to QB electron transfer to ascertain changes if any. Most of the studies relating to heat stress deal with in vitro experiments using isolated chloroplasts or PSII/PSI sub-membrane fractions. Therefore, it was felt necessary to study stress response of PSII in in vivo.

F0F1-ATP synthase, which is found intrinsic to the membranes of bacteria, chloroplasts and mitochondria, uses proton-motive force to produce ATP from ADP and Pi (for a recent review see (1)). With respect to the chloroplastic ATP synthase the H+/ATP stoichiometry has been shown to be 4 (2–4). In a foregoing paper we presented the kinetic modeling of this enzyme, based on a comprehensive collection of experimental data (5). In the meantime, convincing evidence has been accumulated that during energy transduction the γ-subunit rotates relative to (αβ)3 (6,7). The structural basis for a rotary mechanism of energy transduction has been outlined by Junge et al. (8,9). According to this picture the rotor is composed of the subunit complex γec12, while the stator is made up of the ensemble (αβ)3δb2a (see Fig. 1). Hypothetical models for torque generation by F0 which resemble closely have been presented by several authors (9-11). According to the proposal of Boyer (12) rotation of γ relative to (αβ)3 is assumed to induce the binding-change in F1, which is coupled with the synthesis and release of ATP. In (5) we introduced γ as an elastic element which accumulates rotational energy generated by F0 before it is transferred to F1. Now, we will consider the detailed kinetic modeling of a definitive rotary mechanism of F0F1-ATP synthase. Open image in new window Figure 1. Scheme of rotatory F0F1-ATP synthase

Illuminated thylakoids, like other energy transducing membranes, couple electron transport to ATP synthesis via the formation of an electrochemical proton gradient that is utilized to make ATP. Electron transport is coupled to proton translocation into the thylakoid lumen (proton electrochemical gradient formation), and proton flux down the electrochemical gradient is coupled to ATP synthesis. When the gradient is not utilized, ΔpH increases and electron transport slows down. Previously It was shown that. at a high external pH and a large proton gradient, when no ATP is synthesized, electron transport proceeds at high rates, a phenomenon termed ‘slip (1–3). Nevertheless, when proton flow through the ATP synthase is blocked with compounds known as energy transfer inhibitors, ΔpH will increase further and electron transport will slow down.

Tentoxin, a cyclic tetrapeptide produced by the fungus Alternaria alternata is known to be a unique energy-transfer inhibitor [1]. It inhibits photophosphorylation in a species-specific manner. The effect of tentoxin on ATPase activity of isolated CF1 depends on its concentration; low concentrations of tentoxin ( 10−5 M) stimulate the activity [2]. These results indicate the existence of at least two types of tentoxin binding site on CF1. The tentoxin binding sites on CF1 seem to be located at least on the β-subunit [3]. As each CF1 has three β-subunits, the existence of three binding sites for tentoxin is plausible, and we identified three types of binding site for tentoxin on CF1 from direct binding studies [4]. Comparison of the Kd values of these three sites with the effects of tentoxin concentration on ATPase activity suggests: the binding of one tentoxin inhibits, that of two tentoxins moderately restores, and that of three tentoxins greatly stimulates the ATPase activity of CF1. This paper describes further studies of the kinetic properties of these binding sites.

The exposure of isolated chloroplasts or leaves of higher plants to thermal stress leads to considerable changes in the structural organization of thylakoid membranes and their photosynthetic activities. The changes in chloroplast architecture (1) were accompanied by heat-induced physical dissociation of the “peripheral” light-harvesting chlorophyll a/b complex II (LHC II) from the PSII core complex (2,3). CO2 fixation, O2 evolution and photophosphorylation capability is markedly inhibited after incubation of isolated chloroplasts at temperature above 35°C (4). PSII-mediated electron transport is reported to be particularly susceptible to heat stress (5). In contrast, PSI-mediated electron transport rate is stimulated at temperature above 35°C (5, 6). The heat-induced increase of P700+, the enhancement of spillover and an increase of absorption cross-section of PSI after short heat treatment were also observed (7).

We have shown previously that exposure of wheat plants to the low temperature or high irradiance is accompanied by increase in sucrose/starch ratio, decrease in Glc 6-P/Fru 6-P ratio, triose-P accumulation, decreased ATP/ADP ratio and concomitant limitations of electron transport (1). It has been shown previously that restriction in phosphate recycling and photophosphorylation and an occurrence of feedback limited photosynthesis under low temperature stress and the high irradiance results from inhibition of starch synthesis (2). However, cold acclimated Monopol exhibited a 1.6-fold stimulation of CO2 assimilation rates compared to controls due to the increased capacity of Calvin cycle enzymes and differential capacity to adjust sucrose phosphate synthase activity. In contrast, cold acclimated Katepwa exhibited a reduction in CO2 assimilation rates compared to controls (1). Alternatively, growth at high irradiance resulted in a limited adjustment of Calvin cycle enzymes and SPS activity, but comparable photosynthetic capacity to that of controls and cold acclimated winter wheat plants (3). We have suggested that differential cultivar-specific adjustment of CO2 assimilation rates is related to the differential cultivar-dependent adjustment of carbon partitioning to sucrose and fructan biosynthesis (1).

The regulatory interaction between the primary photosynthetic processes and carbon assimilation is an important part of photosynthetic regulation in general. Some experimental works [1] and the analysis of theoretical models suggest that [NADPH]/[ATP] ratio is a governing parameter in the regulation of CO2 fixation, linking the Calvin cycle up to electron transport chain and photophosphorylation. This ratio can be optimised by electron flow distribution between various pathways of electron transport. While the existence of linear and cyclic electron flow around the PS 1 is experimentally proved, the cyclic electron transport around the PS 2, proposed some time ago [2], is still a hypothesis. As all these electron transport pathways meet on the level of free plastoquinones, the state of plastoquinone pool can be expected to play a key role in regulation, including plant adaptation to changes in light intensity and spectral composition. These adaptational changes are visualised in Fig. 1.

In previous studies in our laboratory, it was found that exchanging CF1 between broad bean (Vicia faba L) and spinach (Spinacia oleracea L) chloroplasts showed enhancement in photophosphorylation activities. When deficient thylakoid membranes (-CF1) from spinach chloroplasts was incubated with coupling factors from broad bean, the restored photophosphorylation activity was much higher than that incubated with spinach CF1 (1). In order to probe into the mechanism of the enhancement, we made some comparative studies on the function and structure of CF1 from different plants. Many phenomena involved the E-subunit of CF1. Now in this paper we compared specially the structure and function of the E-subunit of CF1 from different plant chloroplasts.

Permeant amines are specific uncouplers that inhibit photophosphorylation without any stimulation of electron transport (1-4). It was also found (3, 4) that tertiary amines reduce ΔpH measured with other permeant amines (ΔpH-probes). This seems to be due to a competition between amine cations for the lumenal binding sites (5). Therefore, inhibition of ATP synthesis is not connected with decrease of ΔpH. The mechanism of this process should not be considered as finally established.

ATP synthases perform the central task in chemiosmotic energy conversion during photosynthetic or oxidative phosphorylation of ADP. These enzymes comprise two subcomplexes: the F0 part allows transmembrane transfer of coupling ions, and F1 harbours the nucleotide binding sites of the enzyme. Our work deals with the coupling regions of the complex, i.e. those sites where interaction between both parts occur.

The local anesthetic dibucaine (DC) has been reported to exert unique ‘selective uncoupling’ effects on PSII electron transport, photophosphorylation, the ΔpH, the xanthophyll cycle and PSII energy dissipation (1–3). However, the molecular nature of the putative selective uncoupling was not understood. Here we summarize the first comparative analysis of the effects of related lipophilic tertiary amines, including the ΔpH indicator 9-aminoacridine (9AA) and DC, on the ΔpH, xanthophyll cycle deepoxidation, PSII energy dissipation, electron transport and ATPase activity. The results are discussed with respect to recent ultra-structural and biochemical evidence that lipophilic ΔpH indicators and local anesthetics bind competitively to thylakoid membranes while the local anesthetics also exert dramatic perturbations to the stromal and agranal thylakoid membranes (4, 5).

The mechanism of relation between primary bioenergetics processes of photo-synthesis and high crop production in wheat and it importance for creation of the Optimal Photosynthetic Plant Type (OPPT) Conception as the biotechnological model is not known yet. Spectrophotometric, polarographic and chlorophyll fluorescence methods were used for the determination of primary bioenergetic processes in photosystem II and chloroplasts on natural gene -controlled models of wheat. We have shown the intensification of noncyclic electron transport, total photophosphorylation, light coupling of energetic processes in chloroplast; the increasing of the chlorophyll a contents and the reaction center of photosystem II activity for OPPT with maximum productivity. Consequently, OPPT had genes controlled photosynthetic “strong” photosystem II and chloroplast. Namely the intensification of photosystem II and chloroplast function is an important factor in regulating the rate of photosynthesis CO2 — gas — changes and productivity. The mechanism of endogenous regulation of photosynthetic bioenergetic processes and the role of integrated approach in OPPT are observed.

ATP synthase (FOF1) produces ATP from ADP and Pi at the expense of protonmotive (1) and in some organisms sodiummotive force (2). The chloroplast enzyme translocates four mol/mol protons per ATP from the exoplasmic to the cytoplasmic side of the thylakoid membrane (3;4). Proton transport and ATP-synthesis/hydrolysis are separated in the bipartite structure of the enzyme. The membrane-intrinsic portion, Fo, translocates ions and carries the respective selectivity filter (5;6). When detached from Fo and thereby decoupled from protonmotive driving force Fi catalyzes ATP hydrolysis (F1-ATPase). Depending on the organism, ATP synthase is composed from at least 8 different subunits totalling at least 20 polypeptide chains. The ATP synthase from Escherichia colt serves as a prototype with a subunit composition of (αβ)3γδe for F1 and (Probably) ab2c12 for Fo as schematically illustrated in Fig. 4. The composition and nomenclature for the Fo-portion in cyanobacteria (ab’bc12) and in chloroplasts (IV, I, II, III12) differs from the former one.

Inorganic pyrophosphatases (sPPase; pyrophosphate phosphohydrolase, EC 3.6.1.1) are essential enzymes found in the cytosol and cellular organelles that drive anabolism through the hydrolysis of the energy-nch pyrophosphate (PPi) produced in the synthesis of biological polymers, making these processes thermodinamically irreversible. Since this hydrolysis replenishes Pi to the energy-converting systems, sPPases should play an important role in the intracellular “phosphate cycle”. In plants and some bacteria, sPPase also participates in sulfur metabolism by coupling PPi hydrolysis to sulfate activation steps. Moreover, PPi (like ATP) can be synthesized in photosynthetic bacteria as a result of photophosphorylation by a PPi-synthase [1].

Photosynthetic CO2 fixation requires a stoichiometry of 3 ATP + 2 NADPH per CO2 assimilated, while electron transport produces 1 ATP per 1 NADPH. Thus, in intact chloroplast from higher plants, ATP availability limits the overall efficiency of CO2 assimilation (1). In green algae, an unusually high ATP/ADP ratio has been reported (2). Moreover, CCCP has been found to have no effect on photosynthesis under light saturation, whilst fully accelerating mitochondrial respiration (3) and reducing ATP/ADP ratio (4). We have further investigated this insensitivity of photosynthesis to uncoupling at the level of: oxygen evolution and uptake, ATP/ADP ratio and polyphosphate utilisation in whole cells of Chlamydonionas reinhardtii and Chlorella somkiniana.

The thylakoid protein phosphorylation is suggested to be involved in the redistribution of the excitation energy between photosystem 2 (PS2) and photosystem 1 (PSI) (1) and/or by the change in the primary photoproducts stoichiometry (2, 3). Numerous data were achieved which confirm these hypothesis. However investigations involving the studies of changes of linear and cyclic electron transport rates are rare. Some results are not consistent. Some data reliably suggested that an inhibition of PS2 photoreaction takes place (3–8). Data concerning changes of PSI activity are not numerous and they are not confirmed. Enhancement of NADP and methylviologen (MV) photoreduction was reported (3, 9). There is some evidence about photophosphorylation increase (3, 10). But some authors have reported the absence of PSI activity rise under phosphorylation (11–12). Changes of electron transport rates of whole electron transport chain (ETC) induced by membrane protein phosphorylation were studied in some works and the results were also inconsistent. There are data showing that electron transport of the whole ETC is not changed (10) or it was reduced (6). Jennings and Zucchelli have demonstrated an increase in NADP photoreduction from water in the presence of gramicidin (13). They concluded that oxidised plastoquinone level reached under phosphorylation conditions caused the change of the NADP reduction rate.

Lipids of the thylakoid membrane show, as demonstrated by chemical, enzymatic, immunological and extraction analyses with solvents of differing polarities, an asymetric distribution in the transversal direction of the membrane [1–6]. Moreover, as functional complexes as PSI, PSII, the LHCP-complex, cytochrome b6 and the ATPase complex of photophosphorylation differ with respect to their lipid composition, and as these complexes show lateral heterogenety in their distribution between grana and intergrana, the lipid distribution differs also in the grana and intergrana regions of the membrane system [1, 5, 7–9]. The lipids are adsorbed to these functional complexes by intermolecular interactions of differing strength. Corresponding to these different binding affinities, different functions might be attributed to these lipids. The major portion of these lipids is extractable with acetone and other polar organic solvents. Only a few lipid molecules are tighter bound onto the peptide subunits of these complexes and escape the extraction procedure. Even after SDS-polyacrylamide electrophoretic analyses the different galactolipids, anionic lipids, phospholipids and also carotenoids may still be detected on peptides of these complexes by means of the Western Blot-procedure and the use of monospecific antisera [10–13].

Different species of plants have different resistances to low soil pH. This study was conducted to determine the effect of low pH on the physiological state of shoots and roots, and of energy transformation systems in chloroplasts. The pH of tissues and growth of shoots and roots were measured in pea, wheat and maize plants growing in normal (6.0) and low (3.0) pH solution cultures in a phytotron at 25°C. Light induced proton uptake and photophosphorylation in chloroplasts isolated from pea and maize plants growing at pH 6.0 were measured in vitro at pHs ranging from 6.9 to 8.8 in the reaction medium. Pea was the most resistant to low pH stress and maize the least resistant, whereas wheat took an intermediate position. When grown at pH 3.0, pea roots showed a greater degree of alkalinisation than wheat roots, whereas maize roots became more acidic. Under low pH, pea shoots and wheat shoots initially became more alkaline or remained unchanged, whereas the pH of maize shoots either remained unchanged or became more acidic. However, in time, shoot tissue pH became more acidic in all species. Pea-root tissue was more acidic than pea-shoot tissue, whereas in wheat and maize, the root tissue was more alkaline than the shoot tissue at both normal and low pHs. However, at pH 3.0, the differences between the pH of the shoot tissue and the root tissue were about half those at 6.0. In all species, growth of shoots and roots was retarded at pH 3.0, but the extent of growth depression was greatest in maize, intermediate in wheat and least in pea. ATP formation in chloroplasts of pea was higher than that in maize under normal (7.8) and low pH of the reaction medium. This correlates with a much higher concentration of sulfhydryl compounds in pea chloroplasts, providing functional reliability of energy storage systems. A scheme of regulation of energy transportation in chloroplasts is discussed.

Heavy metals have adverse effects on plant growth and metabolism. Photosynthesis is quite sensitive to heavy metal toxicity and both in vivo ano in vitro photosynthetic CO2 fixation are affected by heavy metals. Most of the elements studied are inhibitory to photosystem (PS) II, PS I being less sensitive in isolated chloroplasts. A common site of action seems to be at the oxidizing side of PS II. Photophosphorylation and/or enzyme activity are also possible sites of inhibition. Inhibition in vivo seems to be due to multiple effects of these metals. The immediate effect is on stomatal closure followed by chloroplastic changes. Long-tenn exposure results in reduced leaf growth, decreased photosynthetic pigments, chlil1ged chloroplast structure, and decreased enzyme activities for CO2 assimilation.

Much of our present knowledge concerning the nature and regulation of processes such as electron transport, photophosphorylation and CO2 assimilation derives from experiments with isolated chloroplasts and thylakoid membranes. Sub-cellular systems, if isolated with due care, continue to catalyse their component reactions at rates which equal those observed in vivo. It is widely accepted that fully functional organelles provide a more realistic basis for such studies than those which have been unintentionally stripped of their limiting envelopes during isolation.

The H+-ATP synthase of mitochondria is the key enzyme of energy metabolism in eukaryotic cells (Senior, 1988). It is responsible for the synthesis of ATP supported by respiration, which covers, under normal conditions, more than 80% of the energy demand of mammalian cells. An adult man with a daily energy demand of some 3000 kcal turns over some 400 mol of ATP (200 kg). The brain is the organ with the highest demand for aerobic ATP, accounting, with only 2% of the body weight, for 20% of total oxygen consumption at rest (Erecinksa and Silver, 1989).

Antibodies, by reacting with some C-terminal residues of CF1 δ, accessible and thus exposed in situ on the thylakoid membrane, inhibit the PMS mediated cyclic photophosphorylation.

A low-potential c-type cytochrome occurs in a number of blue-green algae [1–4]. The cytochrome contains one mesoheme [5] per 15500 Da polypeptide [3]. The function of the cytochrome is not known, but there is evidence suggesting a role in cyclic photophosphorylation [6].