Absence of alka(e)nes triggers profound remodeling of glycerolipid and carotenoid composition in cyanobacteria membrane.

Alka(e)nes are produced by many living organisms and exhibit diverse physiological roles, reflecting a high functional versatility. Alka(e)nes serve as waterproof wax in plants, communicating pheromones for insects, and microbial signaling molecules in some bacteria. Although alka(e)nes have been found in cyanobacteria and algal chloroplasts, their importance for photosynthetic membranes has remained elusive. In this study, we investigated the consequences of the absence of alka(e)nes on membrane lipid composition and photosynthesis using the cyanobacterium Synechocystis PCC6803 as a model organism. By following the dynamics of membrane lipids and the photosynthetic performance in strains defected and altered in alka(e)ne biosynthesis, we show that drastic changes in the glycerolipid contents occur in the absence of alka(e)nes, including a decrease in the membrane carotenoid content, a decrease in some digalactosyldiacylglycerol (DGDG) species and a parallel increase in monogalactosyldiacylglycerol (MGDG) species. These changes are associated with a higher susceptibility of photosynthesis and growth to high light in alka(e)ne-deficient strains. All these phenotypes are reversed by expressing an algal photoenzyme producing alka(e)nes from fatty acids. Therefore, alkenes, despite their low abundance, are an essential component of the lipid composition of membranes. The profound remodeling of lipid composition that results from their absence suggests that they play an important role in one or more membrane properties in cyanobacteria. Moreover, the lipid compensatory mechanism observed is not sufficient to restore normal functioning of the photosynthetic membranes, particularly under high light intensity. We conclude that alka(e)nes play a crucial role in maintaining lipid homeostasis of thylakoid membranes, thereby contributing to the proper functioning of photosynthesis, particularly under elevated light intensities.

Alternative electron pathways of photosynthesis power green algal CO2 capture.

Microalgae contribute to about half of global net photosynthesis, which converts sunlight into the chemical energy (ATP and NADPH) used to transform CO2 into biomass. Alternative electron pathways of photosynthesis have been proposed to generate additional ATP that is required to sustain CO2 fixation. However, the relative importance of each alternative pathway remains elusive. Here, we dissect and quantify the contribution of cyclic, pseudo-cyclic and chloroplast-to-mitochondria electron flows for their ability to sustain net photosynthesis in the microalga Chlamydomonas reinhardtii. We show that (i) each alternative pathway can provide sufficient additional energy to sustain high CO2 fixation rates, (ii) the alternative pathways exhibit cross-compensation, and (iii) the activity of at least one of the three alternative pathways is necessary to sustain photosynthesis. We further show that all pathways have very different efficiencies at energizing CO2 fixation, with the chloroplast-mitochondria interaction being the most efficient. Overall, our data lay bioenergetic foundations for biotechnological strategies to improve CO2 capture and fixation.

The Phytophthora parasitica effector AVH195 interacts with ATG8, attenuates host autophagy, and promotes biotrophic infection.

BACKGROUND: Plant pathogens secrete effector proteins into host cells to suppress immune responses and manipulate fundamental cellular processes. One of these processes is autophagy, an essential recycling mechanism in eukaryotic cells that coordinates the turnover of cellular components and contributes to the decision on cell death or survival. RESULTS: We report the characterization of AVH195, an effector from the broad-spectrum oomycete plant pathogen, Phytophthora parasitica. We show that P. parasitica expresses AVH195 during the biotrophic phase of plant infection, i.e., the initial phase in which host cells are maintained alive. In tobacco, the effector prevents the initiation of cell death, which is caused by two pathogen-derived effectors and the proapoptotic BAX protein. AVH195 associates with the plant vacuolar membrane system and interacts with Autophagy-related protein 8 (ATG8) isoforms/paralogs. When expressed in cells from the green alga, Chlamydomonas reinhardtii, the effector delays vacuolar fusion and cargo turnover upon stimulation of autophagy, but does not affect algal viability. In Arabidopsis thaliana, AVH195 delays the turnover of ATG8 from endomembranes and promotes plant susceptibility to P. parasitica and the obligate biotrophic oomycete pathogen Hyaloperonospora arabidopsidis. CONCLUSIONS: Taken together, our observations suggest that AVH195 targets ATG8 to attenuate autophagy and prevent associated host cell death, thereby favoring biotrophy during the early stages of the infection process.

The role of BST4 in the pyrenoid of Chlamydomonas reinhardtii.

In many eukaryotic algae, CO2 fixation by Rubisco is enhanced by a CO2-concentrating mechanism, which utilizes a Rubisco-rich organelle called the pyrenoid. The pyrenoid is traversed by a network of thylakoid-membranes called pyrenoid tubules, proposed to deliver CO2. In the model alga Chlamydomonas reinhardtii (Chlamydomonas), the pyrenoid tubules have been proposed to be tethered to the Rubisco matrix by a bestrophin-like transmembrane protein, BST4. Here, we show that BST4 forms a complex that localizes to the pyrenoid tubules. A Chlamydomonas mutant impaired in the accumulation of BST4 (bst4) formed normal pyrenoid tubules and heterologous expression of BST4 in Arabidopsis thaliana did not lead to the incorporation of thylakoids into a reconstituted Rubisco condensate. Chlamydomonas bst4 mutant did not show impaired growth at air level CO2. By quantifying the non-photochemical quenching (NPQ) of chlorophyll fluorescence, we show that bst4 displays a transiently lower thylakoid lumenal pH during dark to light transition compared to control strains. When acclimated to high light, bst4 had sustained higher NPQ and elevated levels of light-induced H2O2 production. We conclude that BST4 is not a tethering protein, but rather is an ion channel involved in lumenal pH regulation possibly by mediating bicarbonate transport across the pyrenoid tubules.

CRISPR interference screens reveal growth-robustness tradeoffs in Synechocystis sp. PCC 6803 across growth conditions.

Barcoded mutant libraries are a powerful tool for elucidating gene function in microbes, particularly when screened in multiple growth conditions. Here, we screened a pooled CRISPR interference library of the model cyanobacterium Synechocystis sp. PCC 6803 in 11 bioreactor-controlled conditions, spanning multiple light regimes and carbon sources. This gene repression library contained 21,705 individual mutants with high redundancy over all open reading frames and noncoding RNAs. Comparison of the derived gene fitness scores revealed multiple instances of gene repression being beneficial in 1 condition while generally detrimental in others, particularly for genes within light harvesting and conversion, such as antennae components at high light and PSII subunits during photoheterotrophy. Suboptimal regulation of such genes likely represents a tradeoff of reduced growth speed for enhanced robustness to perturbation. The extensive data set assigns condition-specific importance to many previously unannotated genes and suggests additional functions for central metabolic enzymes. Phosphoribulokinase, glyceraldehyde-3-phosphate dehydrogenase, and the small protein CP12 were critical for mixotrophy and photoheterotrophy, which implicates the ternary complex as important for redirecting metabolic flux in these conditions in addition to inactivation of the Calvin cycle in the dark. To predict the potency of sgRNA sequences, we applied machine learning on sgRNA sequences and gene repression data, which showed the importance of C enrichment and T depletion proximal to the PAM site. Fitness data for all genes in all conditions are compiled in an interactive web application.

Energy crosstalk between photosynthesis and the algal CO2-concentrating mechanisms.

Microalgal photosynthesis is responsible for nearly half of the CO2 annually captured by Earth's ecosystems. In aquatic environments where the CO2 availability is low, the CO2-fixing efficiency of microalgae greatly relies on mechanisms - called CO2-concentrating mechanisms (CCMs) - for concentrating CO2 at the catalytic site of the CO2-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). While the transport of inorganic carbon (Ci) across membrane bilayers against a concentration gradient consumes part of the chemical energy generated by photosynthesis, the bioenergetics and cellular mechanisms involved are only beginning to be elucidated. Here, we review the current knowledge relating to the energy requirement of CCMs in the light of recent advances in photosynthesis regulatory mechanisms and the spatial organization of CCM components.

Autocatalytic effect boosts the production of medium-chain hydrocarbons by fatty acid photodecarboxylase.

Ongoing climate change is driving the search for renewable and carbon-neutral alternatives to fossil fuels. Photocatalytic conversion of fatty acids to hydrocarbons by fatty acid photodecarboxylase (FAP) represents a promising route to green fuels. However, the alleged low activity of FAP on C2 to C12 fatty acids seemed to preclude the use for synthesis of gasoline-range hydrocarbons. Here, we reveal that Chlorella variabilis FAP (CvFAP) can convert n-octanoic acid in vitro four times faster than n-hexadecanoic acid, its best substrate reported to date. In vivo, this translates into a CvFAP-based production rate over 10-fold higher for n-heptane than for n-pentadecane. Time-resolved spectroscopy and molecular modeling demonstrate that CvFAP's high catalytic activity on n-octanoic acid is, in part, due to an autocatalytic effect of its n-heptane product, which fills the rest of the binding pocket. These results represent an important step toward a bio-based and light-driven production of gasoline-like hydrocarbons.

Alternative photosynthesis pathways drive the algal CO2-concentrating mechanism.

Global photosynthesis consumes ten times more CO2 than net anthropogenic emissions, and microalgae account for nearly half of this consumption1. The high efficiency of algal photosynthesis relies on a mechanism concentrating CO2 (CCM) at the catalytic site of the carboxylating enzyme RuBisCO, which enhances CO2 fixation2. Although many cellular components involved in the transport and sequestration of inorganic carbon have been identified3,4, how microalgae supply energy to concentrate CO2 against a thermodynamic gradient remains unknown4-6. Here we show that in the green alga Chlamydomonas reinhardtii, the combined action of cyclic electron flow and O2 photoreduction-which depend on PGRL1 and flavodiiron proteins, respectively-generate a low luminal pH that is essential for CCM function. We suggest that luminal protons are used downstream of thylakoid bestrophin-like transporters, probably for the conversion of bicarbonate to CO2. We further establish that an electron flow from chloroplast to mitochondria contributes to energizing non-thylakoid inorganic carbon transporters, probably by supplying ATP. We propose an integrated view of the network supplying energy to the CCM, and describe how algal cells distribute energy from photosynthesis to power different CCM processes. These results suggest a route for the transfer of a functional algal CCM to plants to improve crop productivity.

Physiological functions of malate shuttles in plants and algae.

Subcellular compartmentalization confers evolutionary advantage to eukaryotic cells but entails the need for efficient interorganelle communication. Malate functions as redox carrier and metabolic intermediate. It can be shuttled across membranes through translocators. The interconversion of malate and oxaloacetate mediated by malate dehydrogenases requires oxidation/reduction of NAD(P)H/NAD(P)+; therefore, malate trafficking serves to transport reducing equivalents and this is termed the 'malate shuttle'. Although the term 'malate shuttle' was coined more than 50 years ago, novel functions are still emerging. This review highlights recent findings on the functions of malate shuttles in photorespiration, fatty acid ß-oxidation, interorganelle signaling and its putative role in CO2-concentrating mechanisms. We compare and contrast knowledge in plants and algae, thereby providing an evolutionary perspective on redox trafficking in photosynthetic eukaryotes.

Fatty acid photodecarboxylase is an ancient photoenzyme that forms hydrocarbons in the thylakoids of algae.

Fatty acid photodecarboxylase (FAP) is one of the few enzymes that require light for their catalytic cycle (photoenzymes). FAP was first identified in the microalga Chlorella variabilis NC64A, and belongs to an algae-specific subgroup of the glucose-methanol-choline oxidoreductase family. While the FAP from C. variabilis and its Chlamydomonas reinhardtii homolog CrFAP have demonstrated in vitro activities, their activities and physiological functions have not been studied in vivo. Furthermore, the conservation of FAP activity beyond green microalgae remains hypothetical. Here, using a C. reinhardtii FAP knockout line (fap), we showed that CrFAP is responsible for the formation of 7-heptadecene, the only hydrocarbon of this alga. We further showed that CrFAP was predominantly membrane-associated and that >90% of 7-heptadecene was recovered in the thylakoid fraction. In the fap mutant, photosynthetic activity was not affected under standard growth conditions, but was reduced after cold acclimation when light intensity varied. A phylogenetic analysis that included sequences from Tara Ocean identified almost 200 putative FAPs and indicated that FAP was acquired early after primary endosymbiosis. Within Bikonta, FAP was retained in secondary photosynthetic endosymbiosis lineages but absent from those that lost the plastid. Characterization of recombinant FAPs from various algal genera (Nannochloropsis, Ectocarpus, Galdieria, Chondrus) provided experimental evidence that FAP photochemical activity was present in red and brown algae, and was not limited to unicellular species. These results thus indicate that FAP was conserved during the evolution of most algal lineages where photosynthesis was retained, and suggest that its function is linked to photosynthetic membranes.

Role of an ancient light-harvesting protein of PSI in light absorption and photoprotection.

Diverse algae of the red lineage possess chlorophyll a-binding proteins termed LHCR, comprising the PSI light-harvesting system, which represent an ancient antenna form that evolved in red algae and was acquired through secondary endosymbiosis. However, the function and regulation of LHCR complexes remain obscure. Here we describe isolation of a Nannochloropsis oceanica LHCR mutant, named hlr1, which exhibits a greater tolerance to high-light (HL) stress compared to the wild type. We show that increased tolerance to HL of the mutant can be attributed to alterations in PSI, making it less prone to ROS production, thereby limiting oxidative damage and favoring growth in HL. HLR1 deficiency attenuates PSI light-harvesting capacity and growth of the mutant under light-limiting conditions. We conclude that HLR1, a member of a conserved and broadly distributed clade of LHCR proteins, plays a pivotal role in a dynamic balancing act between photoprotection and efficient light harvesting for photosynthesis.

Membrane Inlet Mass Spectrometry: A Powerful Tool for Algal Research.

Since the first great oxygenation event, photosynthetic microorganisms have continuously shaped the Earth's atmosphere. Studying biological mechanisms involved in the interaction between microalgae and cyanobacteria with the Earth's atmosphere requires the monitoring of gas exchange. Membrane inlet mass spectrometry (MIMS) has been developed in the early 1960s to study gas exchange mechanisms of photosynthetic cells. It has since played an important role in investigating various cellular processes that involve gaseous compounds (O2, CO2, NO, or H2) and in characterizing enzymatic activities in vitro or in vivo. With the development of affordable mass spectrometers, MIMS is gaining wide popularity and is now used by an increasing number of laboratories. However, it still requires an important theory and practical considerations to be used. Here, we provide a practical guide describing the current technical basis of a MIMS setup and the general principles of data processing. We further review how MIMS can be used to study various aspects of algal research and discuss how MIMS will be useful in addressing future scientific challenges.

Algal photosynthesis converts nitric oxide into nitrous oxide.

Nitrous oxide (N2O), a potent greenhouse gas in the atmosphere, is produced mostly from aquatic ecosystems, to which algae substantially contribute. However, mechanisms of N2O production by photosynthetic organisms are poorly described. Here we show that the green microalga Chlamydomonas reinhardtii reduces NO into N2O using the photosynthetic electron transport. Through the study of C. reinhardtii mutants deficient in flavodiiron proteins (FLVs) or in a cytochrome p450 (CYP55), we show that FLVs contribute to NO reduction in the light, while CYP55 operates in the dark. Both pathways are active when NO is produced in vivo during the reduction of nitrites and participate in NO homeostasis. Furthermore, NO reduction by both pathways is restricted to chlorophytes, organisms particularly abundant in ocean N2O-producing hot spots. Our results provide a mechanistic understanding of N2O production in eukaryotic phototrophs and represent an important step toward a comprehensive assessment of greenhouse gas emission by aquatic ecosystems.

Corrigendum: Membrane Inlet Mass Spectrometry: A Powerful Tool for Algal Research.

[This corrects the article DOI: 10.3389/fpls.2020.01302.].

Chlamydomonas cell cycle mutant crcdc5 over-accumulates starch and oil.

The use of algal biomass for biofuel production requires improvements in both biomass productivity and its energy density. Green microalgae store starch and oil as two major forms of carbon reserves. Current strategies to increase the amount of carbon reserves often compromise algal growth. To better understand the cellular mechanisms connecting cell division to carbon storage, we examined starch and oil accumulation in two Chlamydomonas mutants deficient in a gene encoding a homolog of the Arabidopsis Cell Division Cycle 5 (CDC5), a MYB DNA binding protein known to be involved in cell cycle in higher plants. The two crcdc5 mutants (crcdc5-1 and crcdc5-2) were found to accumulate significantly higher amount of starch and oil than their corresponding parental lines. Flow cytometry analysis on synchronized cultures cultivated in a diurnal light/dark cycle revealed an abnormal division of the two mutants, characterized by a prolonged S/M phase, therefore demonstrating its implication in cell cycle in Chlamydomonas. Taken together, these results suggest that the energy saved by a slowdown in cell division is used for the synthesis of reserve compounds. This work highlights the importance in understanding the interplay between cell cycle and starch/oil homeostasis, which should have a critical impact on improving lipid/starch productivity.

Continuous photoproduction of hydrocarbon drop-in fuel by microbial cell factories.

Use of microbes to produce liquid transportation fuels is not yet economically viable. A key point to reduce production costs is the design a cell factory that combines the continuous production of drop-in fuel molecules with the ability to recover products from the cell culture at low cost. Medium-chain hydrocarbons seem ideal targets because they can be produced from abundant fatty acids and, due to their volatility, can be easily collected in gas phase. However, pathways used to produce hydrocarbons from fatty acids require two steps, low efficient enzymes and/or complex electron donors. Recently, a new hydrocarbon-forming route involving a single enzyme called fatty acid photodecarboxylase (FAP) was discovered in microalgae. Here, we show that in illuminated E. coli cultures coexpression of FAP and a medium-chain fatty acid thioesterase results in continuous release of volatile hydrocarbons. Maximum hydrocarbon productivity was reached under low/medium light while higher irradiance resulted in decreased amounts of FAP. It was also found that the production rate of hydrocarbons was constant for at least 5 days and that 30% of total hydrocarbons could be collected in the gas phase of the culture. This work thus demonstrates that the photochemistry of the FAP can be harnessed to design a simple cell factory that continuously produces hydrocarbons easy to recover and in pure form.

Subcellular Energetics and Carbon Storage in Chlamydomonas.

Microalgae have emerged as a promising platform for production of carbon- and energy- rich molecules, notably starch and oil. Establishing an economically viable algal biotechnology sector requires a holistic understanding of algal photosynthesis, physiology, cell cycle and metabolism. Starch/oil productivity is a combined effect of their cellular content and cell division activities. Cell growth, starch and fatty acid synthesis all require carbon building blocks and a source of energy in the form of ATP and NADPH, but with a different requirement in ATP/NADPH ratio. Thus, several cellular mechanisms have been developed by microalgae to balance ATP and NADPH supply which are essentially produced by photosynthesis. Major energy management mechanisms include ATP production by the chloroplast-based cyclic electron flow and NADPH removal by water-water cycles. Furthermore, energetic coupling between chloroplast and other cellular compartments, mitochondria and peroxisome, is increasingly recognized as an important process involved in the chloroplast redox poise. Emerging literature suggests that alterations of energy management pathways affect not only cell fitness and survival, but also influence biomass content and composition. These emerging discoveries are important steps towards diverting algal photosynthetic energy to useful products for biotechnological applications.

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.

Branched-Chain Amino Acid Catabolism Impacts Triacylglycerol Homeostasis in Chlamydomonas reinhardtii.

Nitrogen (N) starvation-induced triacylglycerol (TAG) synthesis, and its complex relationship with starch metabolism in algal cells, has been intensively studied; however, few studies have examined the interaction between amino acid metabolism and TAG biosynthesis. Here, via a forward genetic screen for TAG homeostasis, we isolated a Chlamydomonas (Chlamydomonas reinhardtii) mutant (bkdE1α) that is deficient in the E1α subunit of the branched-chain ketoacid dehydrogenase (BCKDH) complex. Metabolomics analysis revealed a defect in the catabolism of branched-chain amino acids in bkdE1α Furthermore, this mutant accumulated 30% less TAG than the parental strain during N starvation and was compromised in TAG remobilization upon N resupply. Intriguingly, the rate of mitochondrial respiration was 20% to 35% lower in bkdE1α compared with the parental strains. Three additional knockout mutants of the other components of the BCKDH complex exhibited phenotypes similar to that of bkdE1α Transcriptional responses of BCKDH to different N status were consistent with its role in TAG homeostasis. Collectively, these results indicate that branched-chain amino acid catabolism contributes to TAG metabolism by providing carbon precursors and ATP, thus highlighting the complex interplay between distinct subcellular metabolisms for oil storage in green microalgae.