Restricting electron flow at cytochrome b6f when downstream electron acceptors are severely limited.

Photosynthetic organisms frequently experience abiotic stress that restricts their growth and development. Under such circumstances, most absorbed solar energy cannot be used for CO2 fixation and can cause the photoproduction of reactive oxygen species (ROS) that can damage the photosynthetic reaction centers of PSI and PSII, resulting in a decline in primary productivity. This work describes a biological "switch" in the green alga Chlamydomonas reinhardtii that reversibly restricts photosynthetic electron transport (PET) at the cytochrome b6f (Cyt b6f) complex when the capacity for accepting electrons downstream of PSI is severely limited. We specifically show this restriction in STARCHLESS6 (sta6) mutant cells, which cannot synthesize starch when they are limited for nitrogen (growth inhibition) and subjected to a dark-to-light transition. This restriction represents a form of photosynthetic control that causes diminished electron flow to PSI and thereby prevents PSI photodamage but does not appear to rely on a ΔpH. Furthermore, when electron flow is restricted, the plastid alternative oxidase (PTOX) becomes active, functioning as an electron valve that dissipates some excitation energy absorbed by PSII and allows the formation of a proton motive force (PMF) that would drive some ATP production (potentially sustaining PSII repair and nonphotochemical quenching [NPQ]). The restriction at the Cyt b6f complex can be gradually relieved with continued illumination. This study provides insights into how PET responds to a marked reduction in availability of downstream electron acceptors and the protective mechanisms involved.

Alternative outlets for sustaining photosynthetic electron transport during dark-to-light transitions.

Environmental stresses dramatically impact the balance between the production of photosynthetically derived energetic electrons and Calvin-Benson-Bassham cycle (CBBC) activity; an imbalance promotes accumulation of reactive oxygen species and causes cell damage. Hence, photosynthetic organisms have developed several strategies to route electrons toward alternative outlets that allow for storage or harmless dissipation of their energy. In this work, we explore the activities of three essential outlets associated with Chlamydomonas reinhardtii photosynthetic electron transport: (i) reduction of O2 to H2O through flavodiiron proteins (FLVs) and (ii) plastid terminal oxidases (PTOX) and (iii) the synthesis of starch. Real-time measurements of O2 exchange have demonstrated that FLVs immediately engage during dark-to-light transitions, allowing electron transport when the CBBC is not fully activated. Under these conditions, we quantified maximal FLV activity and its overall capacity to direct photosynthetic electrons toward O2 reduction. However, when starch synthesis is compromised, a greater proportion of the electrons is directed toward O2 reduction through both the FLVs and PTOX, suggesting an important role for starch synthesis in priming/regulating CBBC and electron transport. Moreover, partitioning energized electrons between sustainable (starch; energetic electrons are recaptured) and nonsustainable (H2O; energetic electrons are not recaptured) outlets is part of the energy management strategy of photosynthetic organisms that allows them to cope with the fluctuating conditions encountered in nature. Finally, unmasking the repertoire and control of such energetic reactions offers new directions for rational redesign and optimization of photosynthesis to satisfy global demands for food and other resources.

The mitochondrial alternative oxidase from Chlamydomonas reinhardtii enables survival in high light.

Photosynthetic organisms often experience extreme light conditions that can cause hyper-reduction of the chloroplast electron transport chain, resulting in oxidative damage. Accumulating evidence suggests that mitochondrial respiration and chloroplast photosynthesis are coupled when cells are absorbing high levels of excitation energy. This coupling helps protect the cells from hyper-reduction of photosynthetic electron carriers and diminishes the production of reactive oxygen species (ROS). To examine this cooperative protection, here we characterized Chlamydomonas reinhardtii mutants lacking the mitochondrial alternative terminal respiratory oxidases, CrAOX1 and CrAOX2. Using fluorescent fusion proteins, we experimentally demonstrated that both enzymes localize to mitochondria. We also observed that the mutant strains were more sensitive than WT cells to high light under mixotrophic and photoautotrophic conditions, with the aox1 strain being more sensitive than aox2 Additionally, the lack of CrAOX1 increased ROS accumulation, especially in very high light, and damaged the photosynthetic machinery, ultimately resulting in cell death. These findings indicate that the Chlamydomonas AOX proteins can participate in acclimation of C. reinhardtii cells to excess absorbed light energy. They suggest that when photosynthetic electron carriers are highly reduced, a chloroplast-mitochondria coupling allows safe dissipation of photosynthetically derived electrons via the reduction of O2 through AOX (especially AOX1)-dependent mitochondrial respiration.

Bilin-Dependent Photoacclimation in Chlamydomonas reinhardtii.

In land plants, linear tetrapyrrole (bilin)-based phytochrome photosensors optimize photosynthetic light capture by mediating massive reprogramming of gene expression. But, surprisingly, many green algal genomes lack phytochrome genes. Studies of the heme oxygenase mutant (hmox1) of the green alga Chlamydomonas reinhardtii suggest that bilin biosynthesis in plastids is essential for proper regulation of a nuclear gene network implicated in oxygen detoxification during dark-to-light transitions. hmox1 cannot grow photoautotrophically and photoacclimates poorly to increased illumination. We show that these phenotypes are due to reduced accumulation of photosystem I (PSI) reaction centers, the PSI electron acceptors 5'-monohydroxyphylloquinone and phylloquinone, and the loss of PSI and photosystem II antennae complexes during photoacclimation. The hmox1 mutant resembles chlorophyll biosynthesis mutants phenotypically, but can be rescued by exogenous biliverdin IXα, the bilin produced by HMOX1. This rescue is independent of photosynthesis and is strongly dependent on blue light. RNA-seq comparisons of hmox1, genetically complemented hmox1, and chemically rescued hmox1 reveal that tetrapyrrole biosynthesis and known photoreceptor and photosynthesis-related genes are not impacted in the hmox1 mutant at the transcript level. We propose that a bilin-based, blue-light-sensing system within plastids evolved together with a bilin-based retrograde signaling pathway to ensure that a robust photosynthetic apparatus is sustained in light-grown Chlamydomonas.

GreenCut protein CPLD49 of Chlamydomonas reinhardtii associates with thylakoid membranes and is required for cytochrome b6 f complex accumulation.

The GreenCut encompasses a suite of nucleus-encoded proteins with orthologs among green lineage organisms (plants, green algae), but that are absent or poorly conserved in non-photosynthetic/heterotrophic organisms. In Chlamydomonas reinhardtii, CPLD49 (Conserved in Plant Lineage and Diatoms49) is an uncharacterized GreenCut protein that is critical for maintaining normal photosynthetic function. We demonstrate that a cpld49 mutant has impaired photoautotrophic growth under high-light conditions. The mutant exhibits a nearly 90% reduction in the level of the cytochrome b6 f complex (Cytb6 f), which impacts linear and cyclic electron transport, but does not compromise the ability of the strain to perform state transitions. Furthermore, CPLD49 strongly associates with thylakoid membranes where it may be part of a membrane protein complex with another GreenCut protein, CPLD38; a mutant null for CPLD38 also impacts Cytb6 f complex accumulation. We investigated several potential functions of CPLD49, with some suggested by protein homology. Our findings are congruent with the hypothesis that CPLD38 and CPLD49 are part of a novel thylakoid membrane complex that primarily modulates accumulation, but also impacts the activity of the Cytb6 f complex. Based on motifs of CPLD49 and the activities of other CPLD49-like proteins, we suggest a role for this putative dehydrogenase in the synthesis of a lipophilic thylakoid membrane molecule or cofactor that influences the assembly and activity of Cytb6 f.

Gordon Research Conference on photosynthesis: photosynthetic plasticity from the environment to synthetic systems.

Here, we provide a summary of the 2017 Gordon Research Conference on Photosynthesis: "Photosynthetic plasticity: from the environment to synthetic systems". This conference was held at the Grand Summit Resort Hotel at Sunday River, Newry, Maine, USA, from July 16 to 21, 2017. We have also included here a brief description of the Gordon Research Seminar (for students and post-docs) held during 2 days preceding this conference. Following the conclusion of the conference's scientific program, four young scientists (Han Bao, Vivek Tiwari, Setsuko Wakao, and Usha Lingappa) were recognized for their research presentations, each of whom received a book as a gift from one of us (Govindjee). Having chaired the 2015 Gordon Research Conference on Photosynthesis in 2015, Fabrice Rappaport, who lost his fight against cancer in January 2016, was remembered for his profound impact on the field of photosynthesis research.

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.

The Type II NADPH Dehydrogenase Facilitates Cyclic Electron Flow, Energy-Dependent Quenching, and Chlororespiratory Metabolism during Acclimation of Chlamydomonas reinhardtii to Nitrogen Deprivation.

When photosynthetic organisms are deprived of nitrogen (N), the capacity to grow and assimilate carbon becomes limited, causing a decrease in the productive use of absorbed light energy and likely a rise in the cellular reduction state. Although there is a scarcity of N in many terrestrial and aquatic environments, a mechanistic understanding of how photosynthesis adjusts to low-N conditions and the enzymes/activities integral to these adjustments have not been described. In this work, we use biochemical and biophysical analyses of photoautotrophically grown wild-type and mutant strains of Chlamydomonas reinhardtii to determine the integration of electron transport pathways critical for maintaining active photosynthetic complexes even after exposure of cells to N deprivation for 3 d. Key to acclimation is the type II NADPH dehydrogenase, NDA2, which drives cyclic electron flow (CEF), chlororespiration, and the generation of an H(+) gradient across the thylakoid membranes. N deprivation elicited a doubling of the rate of NDA2-dependent CEF, with little contribution from PGR5/PGRL1-dependent CEF The H(+) gradient generated by CEF is essential to sustain nonphotochemical quenching, while an increase in the level of reduced plastoquinone would promote a state transition; both are necessary to down-regulate photosystem II activity. Moreover, stimulation of NDA2-dependent chlororespiration affords additional relief from the elevated reduction state associated with N deprivation through plastid terminal oxidase-dependent water synthesis. Overall, rerouting electrons through the NDA2 catalytic hub in response to photoautotrophic N deprivation sustains cell viability while promoting the dissipation of excess excitation energy through quenching and chlororespiratory processes.

A genome-wide algal mutant library and functional screen identifies genes required for eukaryotic photosynthesis.

Photosynthetic organisms provide food and energy for nearly all life on Earth, yet half of their protein-coding genes remain uncharacterized1,2. Characterization of these genes could be greatly accelerated by new genetic resources for unicellular organisms. Here we generated a genome-wide, indexed library of mapped insertion mutants for the unicellular alga Chlamydomonas reinhardtii. The 62,389 mutants in the library, covering 83% of nuclear protein-coding genes, are available to the community. Each mutant contains unique DNA barcodes, allowing the collection to be screened as a pool. We performed a genome-wide survey of genes required for photosynthesis, which identified 303 candidate genes. Characterization of one of these genes, the conserved predicted phosphatase-encoding gene CPL3, showed that it is important for accumulation of multiple photosynthetic protein complexes. Notably, 21 of the 43 higher-confidence genes are novel, opening new opportunities for advances in understanding of this biogeochemically fundamental process. This library will accelerate the characterization of thousands of genes in algae, plants, and animals.

Nutrient scavenging and energy management: acclimation responses in nitrogen and sulfur deprived Chlamydomonas.

Photosynthetic organisms have evolved to modulate their metabolism to accommodate the highly dynamic light and nutrient conditions in nature. In this review we discuss ways in which the green alga Chlamydomonas reinhardtii acclimates to nitrogen and sulfur deprivation, conditions that would limit the anabolic use of excitation energy because of a markedly reduced capacity for cell growth and division. Major aspects of this acclimation process are stringently regulated and involve scavenging the limited nutrient from internal and external sources, and the redirection of fixed carbon toward energy storage (e.g. starch, oil). However, photosynthetic organisms have also evolved mechanisms to dissipate excess absorbed light energy, and to eliminate potentially dangerous energetic electrons through the reduction of O2 and H+ to H2O; this reduction can occur both through photosynthetic electron transport (e.g. Mehler reaction, chlororespiration) and mitochondrial respiration. Furthermore, algal cells likely exploit other energy management pathways that are currently not linked to nutrient limitation responses or that remain to be identified.

Flocculation of Chlamydomonas reinhardtii with Different Phenotypic Traits by Metal Cations and High pH.

Concentrating algal cells by flocculation as a prelude to centrifugation could significantly reduce the energy and cost of harvesting the algae. However, how variation in phenotypic traits such as cell surface features, cell size and motility alter the efficiency of metal cation and pH-induced flocculation is not well understood. Our results demonstrate that both wild-type and cell wall-deficient strains of the green unicellular alga Chlamydomonas reinhardtii efficiently flocculate (>90%) at an elevated pH of the medium (pH 11) upon the addition of divalent cations such as calcium and magnesium (>5 mM). The trivalent ferric cation (at 10 mM) proved to be essential for promoting flocculation under weak alkaline conditions (pH ∼8.5), with a maximum efficiency that exceeded 95 and 85% for wild-type CC1690 and the cell wall-deficient sta6 mutant, respectively. Near complete flocculation could be achieved using a combination of 5 mM calcium and a pH >11, while the medium recovered following cell removal could be re-cycled without affecting algal growth rates. Moreover, the absence of starch in the cell had little overall impact on flocculation efficiency. These findings contribute to our understanding of flocculation in different Chlamydomonas strains and have implications with respect to inexpensive methods for harvesting algae with different phenotypic traits. Additional research on the conditions (e.g., pH and metal ions) used for efficient flocculation of diverse algal groups with diverse characteristics, at both small and large scale, will help establish inexpensive procedures for harvesting cell biomass.

Structure and flexibility of the C-ring in the electromotor of rotary F(0)F(1)-ATPase of pea chloroplasts.

A ring of 8-15 identical c-subunits is essential for ion-translocation by the rotary electromotor of the ubiquitous F(O)F(1)-ATPase. Here we present the crystal structure at 3.4Šresolution of the c-ring from chloroplasts of a higher plant (Pisum sativum), determined using a native preparation. The crystal structure was found to resemble that of an (ancestral) cyanobacterium. Using elastic network modeling to investigate the ring's eigen-modes, we found five dominant modes of motion that fell into three classes. They revealed the following deformations of the ring: (I) ellipsoidal, (II) opposite twisting of the luminal circular surface of the ring against the stromal surface, and (III) kinking of the hairpin-shaped monomers in the middle, resulting in bending/stretching of the ring. Extension of the elastic network analysis to rings of different c(n)-symmetry revealed the same classes of dominant modes as in P. sativum (c(14)). We suggest the following functional roles for these classes: The first and third classes of modes affect the interaction of the c-ring with its counterparts in F(O), namely subunits a and bb'. These modes are likely to be involved in ion-translocation and torque generation. The second class of deformation, along with deformations of subunits γ and ε might serve to elastically buffer the torque transmission between F(O) and F(1).

The little we know on the structure and machinery of V-ATPase.

The life of every eukaryotic cell depends on the function of vacuolar H(+)-ATPase (V-ATPase). Today we know that V-ATPase is vital for many more physiological and biochemical processes than it was expected three decades ago when the enzyme was discovered. These range from a crucial role in the function of internal organelles such as vacuoles, lysosomes, synaptic vesicles, endosomes, secretory granules and the Golgi apparatus to the plasma membrane of several organisms and specific tissues, and specialized cells. The overall structure and mechanism of action of the V-ATPase is supposed to be similar to that of the well-characterized F-type ATP synthase (F-ATPase). Both consist of a soluble catalytic domain (V(1) or F(1)) that is coupled to a membrane-spanning domain (V(o) or F(o)) by one or more 'stalk' components. Owing to the complexity and challenging properties of V-ATPase its study is lagging behind that of its relative F-ATPase. Time will tell whether V-ATPase shares an identical mechanism of action with F-ATPase or its mode of operation is unique.

Vacuolar H(+)-ATPase-an enzyme for all seasons.

The life of every eukaryotic cell depends on the function of vacuolar H(+)-ATPase (V-ATPase). Because of its complexity and its challenging properties, the study of this enzyme has lagged behind that of its close relative, F-ATPase. We now know that V-ATPase is vital for many more physiological and biochemical processes than anticipated when the enzyme was discovered a few decades ago. It plays a crucial role in the proper functioning of internal organelles such as vacuoles, lysosomes, synaptic vesicles, endosomes, secretory granules, and the Golgi apparatus as well as in plasma membrane of several organisms and specific tissues and specialized cells. Knowledge of its involvement in several diseases, including cancer, has helped to establish the importance of V-ATPase for the preservation of life.