Fabrice Rappaport

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.

Kinetics of electron transfer and electrochromic change during the redox transitions of the photosynthetic oxygen-evolving complex

Abstract The flash-induced kinetics of formation of the successive S-states of the oxygen-evolving complex were analyzed through absorption changes at 295 nm and in the blue region (440-424 nm). The 295 nm change monitors electron transfer from the charge-storing system towards the oxidized tyrosine Y Z . The blue absorption changes are due to a local electrochromic shift that was previously shown to vary in size, both in response to electron transfer and to proton release from the catalytic center. The kinetics of proton release can thus be estimated by comparing the 295 nm and electrochromic responses. The half-times found for electron transfer (at pH 6.5) were 250 μs for Y + Z S 0 → Y Z S 1 , 55 μs for Y + Z S 1 → Y Z S 2 , 290 μs for Y + Z S 2 → Y Z S 3 and 1.2 ms for Y + Z S 3 → Y Z S 0 . The electrochromic kinetics are markedly biphasic during the Y + Z S 3 → Y Z S 0 transition, with a fast phase ( t 1 2 ≈ 30 μ s ) accounting for 40% of the total amplitude and a slow phase ( t 1 2 ≈ 1.2 ms ) concomitant with S 0 and O 2 formation. The fast electrochromic decay is accompanied by a lag in the electron transfer kinetics. This phase is interpreted as reflecting the electrostatically triggered expulsion of one proton from the catalytic center caused by the positive charge on Y + Z . This first step then allows the 1.2 ms reaction to take place. The electrochromic kinetics were also found to be globally faster than electron transfer for the Y + Z S 0 → Y Z S 1 transition above pH 6.5, suggesting similarly a Y + Z -induced deprotonation.

HMA1, a new Cu-ATPase of the chloroplast envelope, is essential for growth under adverse light conditions

Although ions play important roles in the cell and chloroplast metabolism, little is known about ion transport across the chloroplast envelope. Using a proteomic approach specifically targeted to the Arabidopsis chloroplast envelope, we have identified HMA1, which belongs to the metal-transporting P1B-type ATPases family. HMA1 is mainly expressed in green tissues, and we validated its chloroplast envelope localization. Yeast expression experiments demonstrated that HMA1 is involved in copper homeostasis and that deletion of its N-terminal His-domain partially affects the metal transport. Characterization of hma1 Arabidopsis mutants revealed a lower chloroplast copper content and a diminution of the total chloroplast superoxide dismutase activity. No effect was observed on the plastocyanin content in these lines. The hma1 insertional mutants grew like WT plants in standard condition but presented a photosensitivity phenotype under high light. Finally, direct biochemical ATPase assays performed on purified chloroplast envelope membranes showed that the ATPase activity of HMA1 is specifically stimulated by copper. Our results demonstrate that HMA1 offers an additional way to the previously characterized chloroplast envelope Cu-ATPase PAA1 to import copper in the chloroplast.

Back-reactions, short-circuits, leaks and other energy wasteful reactions in biological electron transfer: Redox tuning to survive life in O2

The energy‐converting redox enzymes perform productive reactions efficiently despite the involvement of high energy intermediates in their catalytic cycles. This is achieved by kinetic control: with forward reactions being faster than competing, energy‐wasteful reactions. This requires appropriate cofactor spacing, driving forces and reorganizational energies. These features evolved in ancestral enzymes in a low O2 environment. When O2 appeared, energy‐converting enzymes had to deal with its troublesome chemistry. Various protective mechanisms duly evolved that are not directly related to the enzymes’ principal redox roles. These protective mechanisms involve fine‐tuning of reduction potentials, switching of pathways and the use of short circuits, back‐reactions and side‐paths, all of which compromise efficiency. This energetic loss is worth it since it minimises damage from reactive derivatives of O2 and thus gives the organism a better chance of survival. We examine photosynthetic reaction centres, bc 1 and b 6 f complexes from this view point. In particular, the evolution of the heterodimeric PSI from its homodimeric ancestors is explained as providing a protective back‐reaction pathway. This “sacrifice‐of‐efficiency‐for‐protection” concept should be generally applicable to bioenergetic enzymes in aerobic environments.

Cyclic electron flow is redox-controlled but independent of state transition

Photosynthesis is the biological process that feeds the biosphere with reduced carbon. The assimilation of CO2 requires the fine tuning of two co-existing functional modes: linear electron flow, which provides NADPH and ATP, and cyclic electron flow, which only sustains ATP synthesis. Although the importance of this fine tuning is appreciated, its mechanism remains equivocal. Here we show that cyclic electron flow as well as formation of supercomplexes, thought to contribute to the enhancement of cyclic electron flow, are promoted in reducing conditions with no correlation with the reorganization of the thylakoid membranes associated with the migration of antenna proteins towards Photosystems I or II, a process known as state transition. We show that cyclic electron flow is tuned by the redox power and this provides a mechanistic model applying to the entire green lineage including the vast majority of the cases in which state transition only involves a moderate fraction of the antenna.

An original adaptation of photosynthesis in the marine green alga Ostreococcus

Adaptation of photosynthesis in marine environment has been examined in two strains of the green, picoeukaryote Ostreococcus: OTH95, a surface/high-light strain, and RCC809, a deep-sea/low-light strain. Differences between the two strains include changes in the light-harvesting capacity, which is lower in OTH95, and in the photoprotection capacity, which is enhanced in OTH95. Furthermore, RCC809 has a reduced maximum rate of O2 evolution, which is limited by its decreased photosystem I (PSI) level, a possible adaptation to Fe limitation in the open oceans. This decrease is, however, accompanied by a substantial rerouting of the electron flow to establish an H2O-to-H2O cycle, involving PSII and a potential plastid plastoquinol terminal oxidase. This pathway bypasses electron transfer through the cytochrome b6f complex and allows the pumping of “extra” protons into the thylakoid lumen. By promoting the generation of a large ΔpH, it facilitates ATP synthesis and nonphotochemical quenching when RCC809 cells are exposed to excess excitation energy. We propose that the diversion of electrons to oxygen downstream of PSII, but before PSI, reflects a common and compulsory strategy in marine phytoplankton to bypass the constraints imposed by light and/or nutrient limitation and allow successful colonization of the open-ocean marine environment.

Energetic coupling between plastids and mitochondria drives CO2 assimilation in diatoms

Diatoms are one of the most ecologically successful classes of photosynthetic marine eukaryotes in the contemporary oceans. Over the past 30 million years, they have helped to moderate Earth’s climate by absorbing carbon dioxide from the atmosphere, sequestering it via the biological carbon pump and ultimately burying organic carbon in the lithosphere. The proportion of planetary primary production by diatoms in the modern oceans is roughly equivalent to that of terrestrial rainforests. In photosynthesis, the efficient conversion of carbon dioxide into organic matter requires a tight control of the ATP/NADPH ratio which, in other photosynthetic organisms, relies principally on a range of plastid-localized ATP generating processes. Here we show that diatoms regulate ATP/NADPH through extensive energetic exchanges between plastids and mitochondria. This interaction comprises the re-routing of reducing power generated in the plastid towards mitochondria and the import of mitochondrial ATP into the plastid, and is mandatory for optimized carbon fixation and growth. We propose that the process may have contributed to the ecological success of diatoms in the ocean.

Arabidopsis CURVATURE THYLAKOID1 Proteins Modify Thylakoid Architecture by Inducing Membrane Curvature

Here, we characterize a protein family that forms a complex by oligomerization, which is required for bending of the thylakoid membrane. Without this complex, the typical thylakoid ultrastructure of land plant chloroplasts, composed of grana stacks and stroma lamellae, cannot develop. Strikingly, the loss of this active compartmentalization within thylakoids affects photosynthesis only moderately. Chloroplasts of land plants characteristically contain grana, cylindrical stacks of thylakoid membranes. A granum consists of a core of appressed membranes, two stroma-exposed end membranes, and margins, which connect pairs of grana membranes at their lumenal sides. Multiple forces contribute to grana stacking, but it is not known how the extreme curvature at margins is generated and maintained. We report the identification of the CURVATURE THYLAKOID1 (CURT1) protein family, conserved in plants and cyanobacteria. The four Arabidopsis thaliana CURT1 proteins (CURT1A, B, C, and D) oligomerize and are highly enriched at grana margins. Grana architecture is correlated with the CURT1 protein level, ranging from flat lobe-like thylakoids with considerably fewer grana margins in plants without CURT1 proteins to an increased number of membrane layers (and margins) in grana at the expense of grana diameter in overexpressors of CURT1A. The endogenous CURT1 protein in the cyanobacterium Synechocystis sp PCC6803 can be partially replaced by its Arabidopsis counterpart, indicating that the function of CURT1 proteins is evolutionary conserved. In vitro, Arabidopsis CURT1A proteins oligomerize and induce tubulation of liposomes, implying that CURT1 proteins suffice to induce membrane curvature. We therefore propose that CURT1 proteins modify thylakoid architecture by inducing membrane curvature at grana margins.

Bacteriorhodopsin/Amphipol Complexes: Structural and Functional Properties

The membrane protein bacteriorhodopsin (BR) can be kept soluble in its native state for months in the absence of detergent by amphipol (APol) A8-35, an amphiphilic polymer. After an actinic flash, A8-35-complexed BR undergoes a complete photocycle, with kinetics intermediate between that in detergent solution and that in its native membrane. BR/APol complexes form well defined, globular particles comprising a monomer of BR, a complete set of purple membrane lipids, and, in a peripheral distribution, approximately 2 g APol/g BR, arranged in a compact layer. In the absence of free APol, BR/APol particles can autoassociate into small or large ordered fibrils.

Plastid terminal oxidase 2 (PTOX2) is the major oxidase involved in chlororespiration in Chlamydomonas

By homology with the unique plastid terminal oxidase (PTOX) found in plants, two genes encoding oxidases have been found in the Chlamydomonas genome, PTOX1 and PTOX2. Here we report the identification of a knockout mutant of PTOX2. Its molecular and functional characterization demonstrates that it encodes the oxidase most predominantly involved in chlororespiration in this algal species. In this mutant, the plastoquinone pool is constitutively reduced under dark-aerobic conditions, resulting in the mobile light-harvesting complexes being mainly, but reversibly, associated with photosystem I. Accordingly, the ptox2 mutant shows lower fitness than wild type when grown under phototrophic conditions. Single and double mutants devoid of the cytochrome b6f complex and PTOX2 were used to measure the oxidation rates of plastoquinols via PTOX1 and PTOX2. Those lacking both the cytochrome b6f complex and PTOX2 were more sensitive to light than the single mutants lacking either the cytochrome b6f complex or PTOX2, which discloses the role of PTOX2 under extreme conditions where the plastoquinone pool is overreduced. A model for chlororespiration is proposed to relate the electron flow rate through these alternative pathways and the redox state of plastoquinones in the dark. This model suggests that, in green algae and plants, the redox poise results from the balanced accumulation of PTOX and NADPH dehydrogenase.