Giorgio Forti

Involvement of state transitions in the switch between linear and cyclic electron flow in Chlamydomonas reinhardtii

The energetic metabolism of photosynthetic organisms is profoundly influenced by state transitions and cyclic electron flow around photosystem I. The former involve a reversible redistribution of the light‐harvesting antenna between photosystem I and photosystem II and optimize light energy utilization in photosynthesis whereas the latter process modulates the photosynthetic yield. We have used the wild‐type and three mutant strains of the green alga Chlamydomonas reinhardtii—locked in state I (stt7), lacking the photosystem II outer antennae (bf4) or accumulating low amounts of cytochrome b6f complex (A‐AUU)—and measured electron flow though the cytochrome b6f complex, oxygen evolution rates and fluorescence emission during state transitions. The results demonstrate that the transition from state 1 to state 2 induces a switch from linear to cyclic electron flow in this alga and reveal a strict cause–effect relationship between the redistribution of antenna complexes during state transitions and the onset of cyclic electron flow.

State transitions, cyclic and linear electron transport and photophosphorylation in Chlamydomonas reinhardtii

The relationship between state transitions and the kinetic properties of the electron transfer chain has been studied in Chlamydomonas reinhardtii. The same turnover rate of cytochrome f was found in state 1 and 2. However, while DBMIB was inhibitory in both states, DCMU was effective only in state 1. These observations suggest that linear electron transport was active only in state 1, while a cyclic pathway around photosystem (PS) I operated in state 2. The reversible shift from linear to cyclic electron transport was modulated by changes of PSII antenna size, which inactivated the linear pathway, and by oxygen, which inhibited the cyclic one. Attainment of state 2, under anaerobiosis in the dark, was associated with the decline of the ATP/ADP ratio in the cells and the dark reduction of the intersystem carriers. Upon illumination of the cells, the ATP/ADP ratio increased in a few seconds to the aerobic level. Then, several minutes later, the F(m) returned to the state 1 level, and O(2) evolution was reactivated. This suggests that ATP, though required for photosynthesis, is not the rate-limiting factor in the reactivation of photosynthetic O(2) evolution, which is rather controlled by the redox state of the electron carriers.

Regulation of electron transport in microalgae.

Unicellular algae are characterized by an extreme flexibility with respect to their responses to environmental constraints. This flexibility probably explains why microalgae show a very high biomass yield, constitute one of the major contributors to primary productivity in the oceans and are considered a promising choice for biotechnological applications. Flexibility results from a combination of several factors including fast changes in the light-harvesting apparatus and a strong interaction between different metabolic processes (e.g. respiration and photosynthesis), which all take place within the same cell. Microalgae are also capable of modifying their photosynthetic electron flow capacity, by changing its maximum rate and/or by diverting photogenerated electrons towards different sinks depending on their growth status. In this review, we will focus on the occurrence and regulation of alternative electron flows in unicellular algae and compare data obtained in these systems with those available in vascular plants. This article is part of a Special Issue entitled: Regulation of Electron Transport in Chloroplasts.

In Vivo Changes of the Oxidation-Reduction State of NADP and of the ATP/ADP Cellular Ratio Linked to the Photosynthetic Activity in Chlamydomonas reinhardtii

The ATP/ADP and NADP/NADPH ratios have been measured in whole-cell extract of the green alga Chlamydomonas reinhardtii, to understand their availability for CO2 assimilation by the Calvin cycle in vivo. Measurements were performed during the dark-light transition of both aerobic and anaerobic cells, under illumination with saturating or low light intensity. Two different patterns of behavior were observed: (a) In anaerobic cells, during the lag preceding O2 evolution, ATP was synthesized without changes in the NADP/NADPH ratio, consistently with the operation of cyclic electron flow. (b) In aerobiosis, illumination increased the ATP/ADP ratio independently of the intensity used, whereas the amount of NADPH was decreased at limiting photon flux and regained the dark-adapted level under saturating photon flux. Moreover, under these conditions, the addition of low concentrations of uncouplers stimulated photosynthetic O2 evolution. These observations suggest that the photosynthetic generation of reducing equivalents rather than the rate of ATP formation limits the photosynthetic assimilation of CO2 in C. reinhardtii cells. This situation is peculiar to C. reinhardtii, because neither NADPH nor ATP limited this process in plant leaves, as shown by their increase upon illumination in barley (Hordeum vulgare) leaves, independent of light intensity. Experiments are presented and were designed to evaluate the contribution of different physiological processes that might increase the photosynthetic ATP/NADPH ratio—the Mehler reaction, respiratory ATP supply following the transfer of reducing equivalents via the malate/oxaloacetate shuttle, and cyclic electron flow around PSI—to this metabolic situation.

Partition zone penetration by chymotrypsin, and the localization of the chloroplast flavoprotein and Photosystem II

1. Chymotrypsin treatment of chloroplast membranes inactivates Photosystem II. The inactivation is higher when the activity is measured under low intensity actinic light, suggesting that primary photochemistry is preferentially inactivated. 2. Membrane stacking induced by Mg2+ protects Photosystem II against chymotrypsin inactivation. When the membranes are irreversible unstacked by brief treatment with trypsin, Mg2+ protection against chymotrypsin inactivation of Photosystem II is abolished. 3. The kinetics of inactivation by chymotrypsin of Photosystem II indicates that membrane stacking slows down, but does not prevent, the access of chymotrypsin to Photosystem II, which is mostly located within the partition zones. 4. It is concluded that a partition gap exists between stacked membranes of about 45 A, the size of the chymotrypsin molecule. 5. The kinetics of inhibition of the chloroplast flavoprotein, ferredoxin-NADP reductase, bt its specific antibody is not affected by membrane stacking. This indicates that this enzyme is located outside the partition zones.

Purification and properties of cytochrome f from parsley leaves

Abstract A method for the extraction and purification of cytochrome f from parsley leaves is described. Cytochrome f is obtained in a high state of purity. A molecular weight of 245000 is indicated by Sephadex G-200 chromatography. The absorption spectrum of pure cytochrome f in the reduced state shows peaks at 554·5 mμ, 532.8 mμ, 524 mμ, 422 mμ and 330 mμ and a shoulder at 402 mμ. Cytochrome f possesses catalase (EC 1.11.1.6) activity, with a Kat. f value of 4.7 · 104.

Metabolic Flexibility of the Green Alga Chlamydomonas reinhardtii as Revealed by the Link between State Transitions and Cyclic Electron Flow

In this Review we focus on the conversion of linear photosynthetic electron transport from water to NADP to the cyclic pathway around Photosystem I in the green alga Chlamydomonas reinhardtii. We discuss the strict relationship that exists between the changes in pathways of electron transport and state transitions, i.e., the reversible functional association of light harvesting proteins with one of the two photosystems of oxygenic photosynthesis. Such a link has not been reported in the case of other photosynthetic organisms, where the state transitions do not affect the pathway of electron transport. Rather, they provide a tool to optimise the rate of linear flow. We propose a kinetic-structural model that explains the mechanism of this particular relationship in Chlamydomonas, and discuss the advantages that this peculiar situation gives to the energetic metabolism of this alga.

Studies on the photoreduction of ferredoxin and the ferredoxin-NADP reductase flavoprotein by chloroplasts fragments: Effect of pyrophosphate

Abstract The extinction coefficients of the different oxidation states of the chloroplast flavoprotein, ferredoxin-NADP reductase, have been determined in the spectral region from 600 to 400 nm. The photoreduction of the flavoprotein by chloroplast fragments produces the semiquinone, fp · FADH•, and not the fully reduced form of the enzyme, fp · FADH2. The photoreduction of the flavoprotein, and of NADP, is inhibited by inorganic pyrophosphate. This metabolite does not affect the photoreduction of ferredoxin, and therefore it is a specific inhibitor at the level of the oxidation of reduced ferredoxin in the electron transport system of chloroplasts. These observations are discussed in relation to the function of ferredoxin, flavoprotein, and pyrophosphate in photosynthesis.

The role of ascorbic acid in photosynthetic electron transport

Abstract The role of the ascorbate-ascorbate peroxidase system in photosynthetic electron transport was investigated in stroma-free thylakoids. It was found that high rates of light-dependent oxygen evolution are observed in the presence of ascorbate and H2O2, provided that membrane-bound ascorbate peroxidase is active, to produce monodehydroascorbate. It was found that monodehydroascorbate photoreduction is inhibited by DCMU and DBMIB, and that it showed Emerson enhancement. Monodehydroascorbate was shown to compete for electrons with ferredoxin-NADP at the reducing side of Photosystem I. The photoreduction of monodehydroascorbate generated ΔpH across the membranes, and was stimulated by uncouplers. It is concluded that monodehydroascorbate is an electron acceptor at the reducing side of Photosystem I.

Proton-induced grana formation in chloroplasts. Distribution of chlorophyll-protein complexes and Photosystem II photochemistry

Abstract Lowering the pH of the incubation medium to pH 5.4 leads to grana formation morphologically similar to that induced by metal cations. The same phenomenon is observed in EDTA-washed chloroplasts, indicating that it is not due in part to electrostatic ‘masking’ by residual cations associated with the membranes. Digitonin fractionation studies have indicated that the distribution of the major chlorophyll-protein complexes between granal and stromal membrane regions is similar at pH 5.4 in the absence of Mg 2+ , and at pH 7.4 in the presence of Mg 2+ . Chlorophyll fluorescence induction studies have indicated that the primary photochemistry of Photosystem II (PS II) is stimulated by lowering the pH to 5.4, just as it is upon metal cation addition at higher pH values. The failure to observe such an increase at pH 5.4 by measuring electron transport to ferricyanide is attributed to a combination of an inhibition by this pH of electron transport at a site after Q reduction and an increase in the number of PS II centres detached from the plastoquinone pool. We conclude that the stacked configuration of chloroplast membranes leads to increased PS II primary photochemistry, which is most simply explained in terms of a redistribution of excitation energy towards PS II.