Guanosine tetraphosphate (ppGpp) accumulation inhibits chloroplast gene expression and promotes super grana formation in the moss Physcomitrium (Physcomitrella) patens.

The nucleotides guanosine tetraphosphate and pentaphosphate (or (p)ppGpp) are implicated in the regulation of chloroplast function in plants. (p)ppGpp signalling is best understood in the model vascular plant Arabidopsis thaliana in which it acts to regulate plastid gene expression to influence photosynthesis, plant development and immunity. However, little information is known about the conservation or diversity of (p)ppGpp signalling in other land plants. We studied the function of ppGpp in the moss Physcomitrium (previously Physcomitrella) patens using an inducible system for triggering ppGpp accumulation. We used this approach to investigate the effects of ppGpp on chloroplast function, photosynthesis and growth. We demonstrate that ppGpp accumulation causes a dramatic drop in photosynthetic capacity by inhibiting chloroplast gene expression. This was accompanied by the unexpected reorganisation of the thylakoid system into super grana. Surprisingly, these changes did not affect gametophore growth, suggesting that bryophytes and vascular plants may have different tolerances to defects in photosynthesis. Our findings point to the existence of both highly conserved and more specific targets of (p)ppGpp signalling in the land plants that may reflect different growth strategies.

Interactions Between Carbon Metabolism and Photosynthetic Electron Transport in a Chlamydomonas reinhardtii Mutant Without CO2 Fixation by RuBisCO.

A Chlamydomonas reinhardtii RuBisCO-less mutant, ΔrbcL, was used to study carbohydrate metabolism without fixation of atmospheric carbon. The regulatory mechanism(s) that control linear electron flow, known as "photosynthetic control," are amplified in ΔrbcL at the onset of illumination. With the aim to understand the metabolites that control this regulatory response, we have correlated the kinetics of primary carbon metabolites to chlorophyll fluorescence induction curves. We identify that ΔrbcL in the absence of acetate generates adenosine triphosphate (ATP) via photosynthetic electron transfer reactions. Also, metabolites of the Calvin Benson Bassham (CBB) cycle are responsive to the light. Indeed, ribulose 1,5-bisphosphate (RuBP), the last intermediate before carboxylation by Ribulose-1,5-bisphosphate carboxylase-oxygenase, accumulates significantly with time, and CBB cycle intermediates for RuBP regeneration, dihydroxyacetone phosphate (DHAP), pentose phosphates and ribose-5-phosphate (R5P) are rapidly accumulated in the first seconds of illumination, then consumed, showing that although the CBB is blocked, these enzymes are still transiently active. In opposition, in the presence of acetate, consumption of CBB cycle intermediates is strongly diminished, suggesting that the link between light and primary carbon metabolism is almost lost. Phosphorylated hexoses and starch accumulate significantly. We show that acetate uptake results in heterotrophic metabolism dominating phototrophic metabolism, with glyoxylate and tricarboxylic acid (TCA) cycle intermediates being the most highly represented metabolites, specifically succinate and malate. These findings allow us to hypothesize which metabolites and metabolic pathways are relevant to the upregulation of processes like cyclic electron flow that are implicated in photosynthetic control mechanisms.

Plastoquinone homeostasis in plant acclimation to light intensity.

Arabidopsis plants were grown from seeds at different photon flux densities (PFDs) of white light ranging from 65 to 800 µmol photons m-2 s-1. Increasing PFD brought about a marked accumulation of plastoquinone (PQ) in leaves. However, the thylakoid photoactive PQ pool, estimated to about 700 pmol mg-1 leaf dry weight, was independent of PFD; PQ accumulation in high light mostly occurred in the photochemically non-active pool (plastoglobules, chloroplast envelopes) which represented up to 75% of total PQ. The amounts of PSII reaction center (on a leaf dry weight basis) also were little affected by PFD during growth, leading to a constant PQ/PSII ratio at all PFDs. Boosting PQ biosynthesis by overexpression of a solanesyl diphosphate-synthesizing enzyme strongly enhanced the PQ levels, particularly at high PFDs. Again, this accumulation occurred exclusively in the non-photoactive PQ pool. Mutational suppression of the plastoglobular ABC1K1 kinase led to a selective reduction of the thylakoid PQ pool size to ca. 400 pmol mg-1 in a large range of PFDs, which was associated with a restriction of the photosynthetic electron flow. Our results show that photosynthetic acclimation to light intensity does not involve modulation of the thylakoid PQ pool size or the amounts of PSII reaction centers. There appears to be a fixed amount of PQ molecules for optimal interaction with PSII and efficient photosynthesis, with the extra PQ molecules being stored outside the thylakoid membranes, implying a tight regulation of PQ distribution within the chloroplasts.

Standard units for ElectroChromic Shift measurements in plant biology.

The absorbance shift of pigments is proportional to the membrane potential (Δψ) in plants, green algae, and many photosynthetic bacteria. It is currently denoted as ElectroChromic Shift (ECS) at 515-520 nm for plant carotenoids. It is increasingly being used for phenotyping plants for traits related to photosynthesis or chloroplast metabolism because it is a non-invasive technique and also because more instruments are now commercially available from various manufacturers. The ECS technique is currently used to monitor the post-illumination decay of the proton-motive force (pmf), but it has a more general use for quantitative studies on photosynthetic energy transduction. Here we briefly summarize the basic knowledge on ECS, emphasize the full potential of this technique, and propose a quantitative analysis of the photosynthetic performance with the definition of a transmission coefficient for electrons along the photosynthetic chain.

Photosynthetic complexes and light-dependant electron transport chain in the aerobic anoxygenic phototroph Roseicyclus mahoneyensis and its spontaneous mutants.

Spontaneous photosynthetic mutants of the aerobic anoxygenic phototrophic bacterium Roseicyclus mahoneyensis, strain ML6 have been identified based on phenotypic differences and spectrophotometric analysis. ML6 contains a reaction centre (RC) with absorption peaks at 755, 800, and 870 nm, light harvesting (LH) complex 1 at 870 nm, and monomodal LH2 at 805 nm; the mutant ML6(B) has only the LH2; ML6(DB) has also lost the LH1; in ML6(BN9O), the LH2 is absent and concentrations of LH1 and RC are much lower than in the wild type. RCs were isolated and purified from ML6 and ML6(BN9O); LH1-RC from ML6; and LH2 from ML6, ML6(B), and ML6(DB). All protein subunits composing the complexes were found to be of typical size. Flash-induced difference spectra revealed ML6 has a fully functional photosynthetic apparatus under aerobic and microaerophilic conditions, and as is typical for AAP, there is no photosynthetic activity anaerobically. ML6(BN9O), while also functional photosynthetically aerobically, showed lower rates due to the lack of LH2 and decreased concentrations of LH1 and RC. ML6(B) and ML6(DB) showed no photoinduced electron transport. Action spectra of light-mediated reactions were also performed on ML6 and ML6(BN9O) to reveal that the majority of carotenoids are not involved in light harvesting. Finally, redox titrations were carried out on membranes of ML6 and ML6(BN9O) to confirm that midpoint redox potentials of the QA, RC-bound cytochrome, and P+ were similar in both strains. QA midpoint potential is + 65 mV, cytochrome is + 245 mV, and P+ is + 430 mV.

The function of PROTOPORPHYRINOGEN IX OXIDASE in chlorophyll biosynthesis requires oxidised plastoquinone in Chlamydomonas reinhardtii.

In the last common enzymatic step of tetrapyrrole biosynthesis, prior to the branching point leading to the biosynthesis of heme and chlorophyll, protoporphyrinogen IX (Protogen) is oxidised to protoporphyrin IX (Proto) by protoporphyrinogen IX oxidase (PPX). The absence of thylakoid-localised plastid terminal oxidase 2 (PTOX2) and cytochrome b6f complex in the ptox2 petB mutant, results in almost complete reduction of the plastoquinone pool (PQ pool) in light. Here we show that the lack of oxidised PQ impairs PPX function, leading to accumulation and subsequently uncontrolled oxidation of Protogen to non-metabolised Proto. Addition of 3(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU) prevents the over-reduction of the PQ pool in ptox2 petB and decreases Proto accumulation. This observation strongly indicates the need of oxidised PQ as the electron acceptor for the PPX reaction in Chlamydomonas reinhardtii. The PPX-PQ pool interaction is proposed to function as a feedback loop between photosynthetic electron transport and chlorophyll biosynthesis.

Structure-Function Analysis of Chloroplast Proteins via Random Mutagenesis Using Error-Prone PCR.

Site-directed mutagenesis of chloroplast genes was developed three decades ago and has greatly advanced the field of photosynthesis research. Here, we describe a new approach for generating random chloroplast gene mutants that combines error-prone polymerase chain reaction of a gene of interest with chloroplast complementation of the knockout Chlamydomonas reinhardtii mutant. As a proof of concept, we targeted a 300-bp sequence of the petD gene that encodes subunit IV of the thylakoid membrane-bound cytochrome b6f complex. By sequencing chloroplast transformants, we revealed 149 mutations in the 300-bp target petD sequence that resulted in 92 amino acid substitutions in the 100-residue target subunit IV sequence. Our results show that this method is suited to the study of highly hydrophobic, multisubunit, and chloroplast-encoded proteins containing cofactors such as hemes, iron-sulfur clusters, and chlorophyll pigments. Moreover, we show that mutant screening and sequencing can be used to study photosynthetic mechanisms or to probe the mutational robustness of chloroplast-encoded proteins, and we propose that this method is a valuable tool for the directed evolution of enzymes in the chloroplast.

The plastoquinone pool outside the thylakoid membrane serves in plant photoprotection as a reservoir of singlet oxygen scavengers.

The Arabidopsis vte1 mutant is devoid of tocopherol and plastochromanol (PC-8). When exposed to excess light energy, vte1 produced more singlet oxygen (1 O2 ) and suffered from extensive oxidative damage compared with the wild type. Here, we show that overexpressing the solanesyl diphosphate synthase 1 (SPS1) gene in vte1 induced a marked accumulation of total plastoquinone (PQ-9) and rendered the vte1 SPS1oex plants tolerant to photooxidative stress, indicating that PQ-9 can replace tocopherol and PC-8 in photoprotection. High total PQ-9 levels were associated with a noticeable decrease in 1 O2 production and higher levels of Hydroxyplastoquinone (PQ-C), a 1 O2 -specific PQ-9 oxidation product. The extra PQ-9 molecules in the vte1 SPS1oex plants were stored in the plastoglobules and the chloroplast envelopes, rather than in the thylakoid membranes, whereas PQ-C was found almost exclusively in the thylakoid membranes. Upon exposure of wild-type plants to high light, the thylakoid PQ-9 pool decreased, whereas the extrathylakoid pool remained unchanged. In vte1 and vte1 SPS1oex plants, the PQ-9 losses in high light were strongly amplified, affecting also the extrathylakoid pool, and PQ-C was found in high amounts in the thylakoids. We conclude that the thylakoid PQ-9 pool acts as a 1 O2 scavenger and is replenished from the extrathylakoid stock.

A stromal region of cytochrome b6f subunit IV is involved in the activation of the Stt7 kinase in Chlamydomonas.

The cytochrome (cyt) b6f complex and Stt7 kinase regulate the antenna sizes of photosystems I and II through state transitions, which are mediated by a reversible phosphorylation of light harvesting complexes II, depending on the redox state of the plastoquinone pool. When the pool is reduced, the cyt b6f activates the Stt7 kinase through a mechanism that is still poorly understood. After random mutagenesis of the chloroplast petD gene, coding for subunit IV of the cyt b6f complex, and complementation of a ΔpetD host strain by chloroplast transformation, we screened for impaired state transitions in vivo by chlorophyll fluorescence imaging. We show that residues Asn122, Tyr124, and Arg125 in the stromal loop linking helices F and G of cyt b6f subunit IV are crucial for state transitions. In vitro reconstitution experiments with purified cyt b6f and recombinant Stt7 kinase domain show that cyt b6f enhances Stt7 autophosphorylation and that the Arg125 residue is directly involved in this process. The peripheral stromal structure of the cyt b6f complex had, until now, no reported function. Evidence is now provided of a direct interaction with Stt7 on the stromal side of the membrane.

Alternative electron transport pathways in photosynthesis: a confluence of regulation.

Photosynthetic reactions proceed along a linear electron transfer chain linking water oxidation at photosystem II (PSII) to CO2 reduction in the Calvin-Benson-Bassham cycle. Alternative pathways poise the electron carriers along the chain in response to changing light, temperature and CO2 inputs, under prolonged hydration stress and during development. We describe recent literature that reports the physiological functions of new molecular players. Such highlights include the flavodiiron proteins and their important role in the green lineage. The parsing of the proton-motive force between ΔpH and Δψ, regulated in many different ways (cyclic electron flow, ATPsynthase conductivity, ion/H+ transporters), is comprehensively reported. This review focuses on an integrated description of alternative electron transfer pathways and how they contribute to photosynthetic productivity in the context of plant fitness to the environment.

Cytochrome b 6 f function and localization, phosphorylation state of thylakoid membrane proteins and consequences on cyclic electron flow.

Both the structure and the protein composition of thylakoid membranes have an impact on light harvesting and electron transfer in the photosynthetic chain. Thylakoid membranes form stacks and lamellae where photosystem II and photosystem I localize, respectively. Light-harvesting complexes II can be associated to either PSII or PSI depending on the redox state of the plastoquinone pool, and their distribution is governed by state transitions. Upon state transitions, the thylakoid ultrastructure and lateral distribution of proteins along the membrane are subject to significant rearrangements. In addition, quinone diffusion is limited to membrane microdomains and the cytochrome b 6 f complex localizes either to PSII-containing grana stacks or PSI-containing stroma lamellae. Here, we discuss possible similarities or differences between green algae and C3 plants on the functional consequences of such heterogeneities in the photosynthetic electron transport chain and propose a model in which quinones, accepting electrons either from PSII (linear flow) or NDH/PGR pathways (cyclic flow), represent a crucial control point. Our aim is to give an integrated description of these processes and discuss their potential roles in the balance between linear and cyclic electron flows.

Critical role of Chlamydomonas reinhardtii ferredoxin-5 in maintaining membrane structure and dark metabolism.

Photosynthetic microorganisms typically have multiple isoforms of the electron transfer protein ferredoxin, although we know little about their exact functions. Surprisingly, a Chlamydomonas reinhardtii mutant null for the ferredoxin-5 gene (FDX5) completely ceased growth in the dark, with both photosynthetic and respiratory functions severely compromised; growth in the light was unaffected. Thylakoid membranes in dark-maintained fdx5 mutant cells became severely disorganized concomitant with a marked decrease in the ratio of monogalactosyldiacylglycerol to digalactosyldiacylglycerol, major lipids in photosynthetic membranes, and the accumulation of triacylglycerol. Furthermore, FDX5 was shown to physically interact with the fatty acid desaturases CrΔ4FAD and CrFAD6, likely donating electrons for the desaturation of fatty acids that stabilize monogalactosyldiacylglycerol. Our results suggest that in photosynthetic organisms, specific redox reactions sustain dark metabolism, with little impact on daytime growth, likely reflecting the tailoring of electron carriers to unique intracellular metabolic circuits under these two very distinct redox conditions.

Redesigning photosynthesis to sustainably meet global food and bioenergy demand.

The world's crop productivity is stagnating whereas population growth, rising affluence, and mandates for biofuels put increasing demands on agriculture. Meanwhile, demand for increasing cropland competes with equally crucial global sustainability and environmental protection needs. Addressing this looming agricultural crisis will be one of our greatest scientific challenges in the coming decades, and success will require substantial improvements at many levels. We assert that increasing the efficiency and productivity of photosynthesis in crop plants will be essential if this grand challenge is to be met. Here, we explore an array of prospective redesigns of plant systems at various scales, all aimed at increasing crop yields through improved photosynthetic efficiency and performance. Prospects range from straightforward alterations, already supported by preliminary evidence of feasibility, to substantial redesigns that are currently only conceptual, but that may be enabled by new developments in synthetic biology. Although some proposed redesigns are certain to face obstacles that will require alternate routes, the efforts should lead to new discoveries and technical advances with important impacts on the global problem of crop productivity and bioenergy production.

Combined increases in mitochondrial cooperation and oxygen photoreduction compensate for deficiency in cyclic electron flow in Chlamydomonas reinhardtii.

During oxygenic photosynthesis, metabolic reactions of CO2 fixation require more ATP than is supplied by the linear electron flow operating from photosystem II to photosystem I (PSI). Different mechanisms, such as cyclic electron flow (CEF) around PSI, have been proposed to participate in reequilibrating the ATP/NADPH balance. To determine the contribution of CEF to microalgal biomass productivity, here, we studied photosynthesis and growth performances of a knockout Chlamydomonas reinhardtii mutant (pgrl1) deficient in PROTON GRADIENT REGULATION LIKE1 (PGRL1)-mediated CEF. Steady state biomass productivity of the pgrl1 mutant, measured in photobioreactors operated as turbidostats, was similar to its wild-type progenitor under a wide range of illumination and CO2 concentrations. Several changes were observed in pgrl1, including higher sensitivity of photosynthesis to mitochondrial inhibitors, increased light-dependent O2 uptake, and increased amounts of flavodiiron (FLV) proteins. We conclude that a combination of mitochondrial cooperation and oxygen photoreduction downstream of PSI (Mehler reactions) supplies extra ATP for photosynthesis in the pgrl1 mutant, resulting in normal biomass productivity under steady state conditions. The lower biomass productivity observed in the pgrl1 mutant in fluctuating light is attributed to an inability of compensation mechanisms to respond to a rapid increase in ATP demand.

Proton gradient regulation 5-mediated cyclic electron flow under ATP- or redox-limited conditions: a study of ΔATpase pgr5 and ΔrbcL pgr5 mutants in the green alga Chlamydomonas reinhardtii.

The Chlamydomonas reinhardtii proton gradient regulation5 (Crpgr5) mutant shows phenotypic and functional traits similar to mutants in the Arabidopsis (Arabidopsis thaliana) ortholog, Atpgr5, providing strong evidence for conservation of PGR5-mediated cyclic electron flow (CEF). Comparing the Crpgr5 mutant with the wild type, we discriminate two pathways for CEF and determine their maximum electron flow rates. The PGR5/proton gradient regulation-like1 (PGRL1) ferredoxin (Fd) pathway, involved in recycling excess reductant to increase ATP synthesis, may be controlled by extreme photosystem I acceptor side limitation or ATP depletion. Here, we show that PGR5/PGRL1-Fd CEF functions in accordance with an ATP/redox control model. In the absence of Rubisco and PGR5, a sustained electron flow is maintained with molecular oxygen instead of carbon dioxide serving as the terminal electron acceptor. When photosynthetic control is decreased, compensatory alternative pathways can take the full load of linear electron flow. In the case of the ATP synthase pgr5 double mutant, a decrease in photosensitivity is observed compared with the single ATPase-less mutant that we assign to a decreased proton motive force. Altogether, our results suggest that PGR5/PGRL1-Fd CEF is most required under conditions when Fd becomes overreduced and photosystem I is subjected to photoinhibition. CEF is not a valve; it only recycles electrons, but in doing so, it generates a proton motive force that controls the rate of photosynthesis. The conditions where the PGR5 pathway is most required may vary in photosynthetic organisms like C. reinhardtii from anoxia to high light to limitations imposed at the level of carbon dioxide fixation.

Redox and ATP control of photosynthetic cyclic electron flow in Chlamydomonas reinhardtii: (II) involvement of the PGR5-PGRL1 pathway under anaerobic conditions.

In oxygenic photosynthesis, cyclic electron flow around photosystem I denotes the recycling of electrons from stromal electron carriers (reduced nicotinamide adenine dinucleotide phosphate, NADPH, ferredoxin) towards the plastoquinone pool. Whether or not cyclic electron flow operates similarly in Chlamydomonas and plants has been a matter of debate. Here we would like to emphasize that despite the regulatory or metabolic differences that may exist between green algae and plants, the general mechanism of cyclic electron flow seems conserved across species. The most accurate way to describe cyclic electron flow remains to be a redox equilibration model, while the supramolecular reorganization of the thylakoid membrane (state transitions) has little impact on the maximal rate of cyclic electron flow. The maximum capacity of the cyclic pathways is shown to be around 60 electrons transferred per photosystem per second, which is in Chlamydomonas cells treated with 3(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and placed under anoxic conditions. Part I of this work (aerobic conditions) was published in a previous issue of BBA-Bioenergetics (vol. 1797, pp. 44-51) (Alric et al., 2010).

Central carbon metabolism and electron transport in Chlamydomonas reinhardtii: metabolic constraints for carbon partitioning between oil and starch.

The metabolism of microalgae is so flexible that it is not an easy task to give a comprehensive description of the interplay between the various metabolic pathways. There are, however, constraints that govern central carbon metabolism in Chlamydomonas reinhardtii that are revealed by the compartmentalization and regulation of the pathways and their relation to key cellular processes such as cell motility, division, carbon uptake and partitioning, external and internal rhythms, and nutrient stress. Both photosynthetic and mitochondrial electron transfer provide energy for metabolic processes and how energy transfer impacts metabolism and vice versa is a means of exploring the regulation and function of these pathways. A key example is the specific chloroplast localization of glycolysis/gluconeogenesis and how it impacts the redox poise and ATP budget of the plastid in the dark. To compare starch and lipids as carbon reserves, their value can be calculated in terms of NAD(P)H and ATP. As microalgae are now considered a potential renewable feedstock, we examine current work on the subject and also explore the possibility of rerouting metabolism toward lipid production.

Inhibition of CO2 fixation by iodoacetamide stimulates cyclic electron flow and non-photochemical quenching upon far-red illumination.

The Benson-Calvin cycle enzymes are activated in vivo when disulfide bonds are opened by reduction via the ferredoxin-thioredoxin system in chloroplasts. Iodoacetamide reacts irreversibly with free -SH groups of cysteine residues and inhibits the enzymes responsible for CO2 fixation. Here, we investigate the effect of iodoacetamide on electron transport, when infiltrated into spinach leaves. Using fluorescence and absorption spectroscopy, we show that (i) iodoacetamide very efficiently blocks linear electron flow upon illumination of both photosystems (decrease in the photochemical yield of photosystem II) and (ii) iodoacetamide favors cyclic electron flow upon light excitation specific to PSI. These effects account for an NPQ formation even faster in iodoacetamide under far-red illumination than in the control under saturating light. Such an increase in NPQ is dependent upon the proton gradient across the thylakoid membrane (uncoupled by nigericin addition) and PGR5 (absent in Arabidopsis pgr5 mutant). Iodoacetamide very tightly insulates the electron current at the level of the thylakoid membrane from any electron leaks toward carbon metabolism, therefore, providing choice conditions for the study of cyclic electron flow around PSI.

Novel thylakoid membrane GreenCut protein CPLD38 impacts accumulation of the cytochrome b6f complex and associated regulatory processes.

Based on previous comparative genomic analyses, a set of nearly 600 polypeptides was identified that is present in green algae and flowering and nonflowering plants but is not present (or is highly diverged) in nonphotosynthetic organisms. The gene encoding one of these "GreenCut" proteins, CPLD38, is in the same operon as ndhL in most cyanobacteria; the NdhL protein is part of a complex essential for cyanobacterial respiration. A cpld38 mutant of Chlamydomonas reinhardtii does not grow on minimal medium, is high light-sensitive under photoheterotrophic conditions, has lower accumulation of photosynthetic complexes, reduced photosynthetic electron flow to P700(+), and reduced photochemical efficiency of photosystem II (ΦPSII); these phenotypes are rescued by a wild-type copy of CPLD38. Single turnover flash experiments and biochemical analyses demonstrated that cytochrome b6f function was severely compromised, and the levels of transcripts and polypeptide subunits of the cytochrome b6f complex were also significantly lower in the cpld38 mutant. Furthermore, subunits of the cytochrome b6f complex in mutant cells turned over much more rapidly than in wild-type cells. Interestingly, PTOX2 and NDA2, two major proteins involved in chlororespiration, were more than 5-fold higher in mutants relative to wild-type cells, suggesting a shift in the cpld38 mutant from photosynthesis toward chlororespiratory metabolism, which is supported by experiments that quantify the reduction state of the plastoquinone pool. Together, these findings support the hypothesis that CPLD38 impacts the stability of the cytochrome b6f complex and possibly plays a role in balancing redox inputs to the quinone pool from photosynthesis and chlororespiration.