Francis-André Wollman

The Dynamics of Photosynthesis

Despite recent elucidation of the three-dimensional structure of major photosynthetic complexes, our understanding of light energy conversion in plant chloroplasts and microalgae under physiological conditions requires exploring the dynamics of photosynthesis. The photosynthetic apparatus is a flexible molecular machine that can acclimate to metabolic and light fluctuations in a matter of seconds and minutes. On a longer time scale, changes in environmental cues trigger acclimation responses that elicit intracellular signaling between the nucleo-cytosol and chloroplast resulting in modification of the biogenesis of the photosynthetic machinery. Here we attempt to integrate well-established knowledge on the functional flexibility of light-harvesting and electron transfer processes, which has greatly benefited from genetic approaches, with data derived from the wealth of recent transcriptomic and proteomic studies of acclimation responses in photosynthetic eukaroytes.

State transitions reveal the dynamics and flexibility of the photosynthetic apparatus

The chloroplast‐based photosynthetic apparatus of plants and algae associates various redox cofactors and pigments with ∼70 polypeptides to form five major transmembrane protein complexes. Among these are two photosystems that have distinct light absorption properties but work in series to produce reducing equivalents aimed at the fixation of atmospheric carbon. A short term chromatic adaptation known as ‘State transitions’ was discovered thirty years ago that allows photosynthetic organisms to adapt to changes in light quality and intensity which would otherwise compromise the efficiency of photosynthetic energy conversion. A two‐decade research effort has finally unraveled the major aspects of the molecular mechanism responsible for State transitions, and their physiological significance has been revisited. This review describes how a—still elusive—regulatory kinase senses the physiological state of the photosynthetic cell and triggers an extensive supramolecular reorganization of the photosynthetic membranes. The resulting picture of the photosynthetic apparatus is that of a highly flexible energy convertor that adapts to the ever‐changing intracellular demand for ATP and/or reducing power.

The Qo site of cytochrome b6f complexes controls the activation of the LHCII kinase.

We created a Qo pocket mutant by site‐directed mutagenesis of the chloroplast petD gene in Chlamydomonas reinhardtii. We mutated the conserved PEWY sequence in the EF loop of subunit IV into PWYE. The pwye mutant did not grow in phototrophic conditions although it assembled wild‐type levels of cytochrome b6f complexes. We demonstrated a complete block in electron transfer through the cytochrome b6f complex and a loss of plastoquinol binding at Qo. The accumulation of cytochrome b6f complexes lacking affinity for plastoquinol enabled us to investigate the role of plastoquinol binding at Qo in the activation of the light‐harvesting complex II (LHCII) kinase during state transitions. We detected no fluorescence quenching at room temperature in state II conditions relative to that in state I. The quantum yield spectrum of photosystem I charge separation in the two state conditions displayed a trough in the absorption region of the major chlorophyll a/b proteins, demonstrating that the cells remained locked in state I. 33Pi labeling of the phosphoproteins in vivo demonstrated that the antenna proteins remained poorly phosphorylated in both state conditions. Thus, the absence of state transitions in the pwye mutant demonstrates directly that plastoquinol binding in the Qo pocket is required for LHCII kinase activation.

A Chloroplast-Targeted Heat Shock Protein 70 (HSP70) Contributes to the Photoprotection and Repair of Photosystem II during and after Photoinhibition

Dark-grown Chlamydomonas reinhardtii cultures that were illuminated at low fluence rates before exposure to high-light conditions exhibited a faster rate of recovery from photoinhibition than did dark-grown cells that were directly exposed to photoinhibitory conditions. This pretreatment has been shown to induce the expression of several nuclear heat shock protein 70 (HSP70) genes, including HSP70B, encoding a chloroplast-localized chaperone. To investigate a possible role of plastidic HSP70B in photoprotection and repair of photosystem II, which is the major target of photoinhibition, we have constructed strains overexpressing or underexpressing HSP70B. The effect of light stress on photosystem II in nuclear transformants harboring HSP70B in the sense or antisense orientation was monitored by measuring variable fluorescence, flash-induced charge separation, and relative amounts of various photosystem II polypeptides. Underexpression of HSP70B caused an increased light sensitivity of photosystem II, whereas overexpression of HSP70B had a protective effect. Furthermore, the reactivation of photosystem II after photoinhibition was enhanced in the HSP70B-overexpressing strain when compared with the wild type, both in the presence or absence of synthesis of chloroplast-encoded proteins. Therefore, HSP70B may participate in vivo both in the molecular protection of the photosystem II reaction centers during photoinhibition and in the process of photosystem II repair.

The biogenesis and assembly of photosynthetic proteins in thylakoid membranes

3. Conveyance of the PPs to the thylakoid membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 3.1. Site of translation of the cyanobacterial and chloroplast-encoded PP subunits . . . . . . . 30 3.2. Targeting to the organelle and import . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 3.3. Maturation of the precursor: the processing step . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 3.4. Insertion, assembly and translocation into the thylakoid membranes . . . . . . . . . . . . . . 34

Changes in light energy distribution upon state transitions: an in vivo photoacoustic study of the wild type and photosynthesis mutants from Chlamydomonas reinhardtii

We investigated, by a photoacoustic method, the changes in light energy distribution to the reaction centres upon state transitions in vivo in Chlamydomonas reinhardtii. We present spectra of the quantum yield of charge separation in the wild type and in several photosynthesis mutants lacking either cytochrome b6f complexes, the PS II cores or the PS I cores. Our results show unambiguously that in the wild-type LHCII becomes connected to PS I in state 2, in a cyt b6f-controlled process. We show that mutants lacking the PS II cores, but not those lacking PS I cores, display a behaviour very similar to that of the wild type upon state transition. Lateral displacement of LHCII, but not of minor antenna complexes (CP26), from the stacked to the unstacked membrane domains results in transfer to PS I of about 80% of the excitation energy absorbed by LHCII. Consequently, in state 2, more than 90% of the chlorophylls transfer energy to PS I in mutants lacking PS II cores. In contrast, a significant proportion of the PS I peripheral antenna is connected with PS II, in state 1 as well as in state 2, in mutants lacking PS I cores. Moreover, in these mutants, phospho-LHCII remains part of PS II antenna in state 2. We attribute these features to the super-stacked organization of the thylakoid membranes in the PS I deficient mutants.

ATP control on state transitions in vivo in Chlamydomonas reinhardtii

Abstract We have attempted to depress the ATP content in dark adapted cells of the unicellular green algae, C. reinhardtii, by inhibiting ATP synthesis coupled to mitochondrial electron flow. Whether we used uncouplers, ATP synthase inhibitors, inhibitors of mitochondrial electron transport or a mutant altered in mitochondrial cytochrome b, we observed a fluorescence quenching at room temperature associated with an increased reduction of the intersystem electron carriers of the photosynthetic apparatus. This fluorescence quenching reflected a genuine transition to state II, since: (i) it was associated with an increase in light-harvesting complex phosphorylation and (ii) it did not occur in mutants lacking the b 6 f complex which are blocked in State I. The complete reversion to state I occurred only into experimental conditions which allowed both ATP synthesis and reoxidation of intersystem electron carriers. We conclude that the intracellular demand for ATP controls state transitions in vivo. The interplay between intracellular ATP concentrations and the redox state of the kinase activator at the thylakoid membrane level is discussed.

A new chlorophyll-protein complex related to Photosystem I in Chlamydomonas reinhardii

Abstract A new chlorophyll-protein complex, CP O, was isolated from Chlamydomonas reinhardii using lithium dodecyl sulfate polyacrylamide gel electrophoresis run at 4°C. A similar complex is recovered using Triton/digitonin solubilization of thylakoid membranes of the F54-14 mutant lacking in CP I and ATPase. CP O is enriched in long-wavelength chlorophyll a and contains five polypeptides (27.5, 27, 25, 23 and 19 kDa). Its 77 K fluorescence emission spectrum peaks at 705 nm while CP II have an emission maximum at 682 and 720 nm, respectively. Comparison of the polypeptide pattern of the wild type and AC40 mutant of C. reinhardii shows that the five CP O polypeptides are specifically lacking in the mutant. Although the 77 K emission originating from the Photosystem (PS) I pigments is lower in the mutant than in the wild type, the two spectra show the same peaks at 686, 694 and 717 nm. However, comparison of the 77 K emission spectrum of the F14 mutant lacking in CP I with that of the double mutant AC40-14 lacking in CP I and CP O shows the absence in the latter of the large emission band peaking at 707 nm. The 707 nm emission is thought to arise from some PS I antennae and is quenched in the wild type by the presence of PS I traps located in CP I. We conclude that CP O is a part of the PS I antenna in C. reinhardii which controls the 707 nm fluorescence emission.

Evidence for a Role of ClpP in the Degradation of the Chloroplast Cytochrome b 6 f Complex

In the green alga Chlamydomonas reinhardtii, the ClpP protease is encoded by an essential chloroplast gene. Mutating its AUG translation initiation codon to AUU reduced ClpP accumulation to 25 to 45% of that of the wild type. Both the mature protein and the putative precursor containing its insertion sequence were present in reduced amounts. Attenuation of ClpP did not affect growth rates under normal conditions but restricted the ability of the cells to adapt to elevated CO2 levels. It also affected the rate of degradation of the cytochrome b6f complex of the thylakoid membrane in two experimental situations: (1) during nitrogen starvation, and (2) in mutants deficient in the Rieske iron–sulfur protein. The ClpP level also controls the steady state accumulation of a mutated version of the Rieske protein. In contrast, attenuation of ClpP did not rescue the fully unassembled subunits in other cytochrome b6f mutants. We conclude that proteolytic disposal of fully or partially assembled cytochrome b6f is controlled by the Clp protease.

Translational regulations as specific traits of chloroplast gene expression.

Studies of protein synthesis in the chloroplast compartment have revealed a unique combination of translational autoregulations and trans‐regulations due to the delivery of a variety of nuclear factors that act post‐transcriptionally. We show how these two characteristics concur to set the major step in the regulation of chloroplast gene expression at the translational level, leading to a surprisingly low sensitivity of chloroplast protein synthesis in response to extensive changes in plastome copy number and transcript concentration.