Search

Microalgae within the Scenedesmaceae are often distinguished by spines, bristles, and other wall characteristics. We examined the dynamic production and chemical nature of bristles extruded from the poles of Tetradesmus deserticola previously isolated from microbiotic crust. Rapidly growing cells in a liquid growth medium were established in polydimethylsiloxane microfluidic chambers specially designed to maintain aerobic conditions over time within a chamber 6–12 μm deep. This geometry enabled in‐focus imaging of single cells over long periods. Differential interference contrast (DIC) imaging revealed that after multiple fission of mother cells, the newly released, lemon‐shaped daughter cells began extruding bristles from each pole. In some instances, the bristles became stuck to either the glass floor or polydimethylsiloxane (PDMS) walls of the chamber, and the force by which the new bristle was extruded was sufficient to propel the cells across the field of view at ~1.2 μm · h−1. Confocal fluorescence and DIC imaging of cells stained with pontamine fast scarlet and calcofluor, and treated with proteinase K, suggested that bristles are proteinaceous and may also host carbohydrate modifications. The polar bristles extruded by this desert‐derived T. deserticola may simply be relics of bristles produced by an aquatic ancestor for flotation or predator deterrence. But, their tendency to attach to glass (silicate) and/or PDMS surfaces suggests a potential role in tethering cells in place or binding soil particles. T. deserticola is closely related to T. obliquus, which is of interest for biofuels development; extruded bristles in T. deserticola may offer tethers for industrial use of these stress‐tolerant algae. [ABSTRACT FROM AUTHOR]

Crustose Verrucariaceae lichens form a distinctive black belt on seashores all over the world. This lifestyle is apparently enabled by a specific set of photobionts. However, their diversity is understudied. We sampled these lichens from the northern Patagonian Pacific coast of Chile. Using molecular markers, we identified both mycobionts and photobionts. The lichens, belonging to the genus Hydropunctaria and to the Wahlenbergiella group, hosted solely Ulvophycean photobionts. Pseudendoclonium submarinum (Kornmanniaceae, Ulvales) was the most common, but representatives of other closely related, yet undescribed, lineages were also found. Undulifilum symbioticum gen. et sp. nov. is described within Kornmanniaceae based on culture morphology and DNA sequence data. Furthermore, the free‐living macroscopic genus Urospora (Acrosiphoniaceae, Ulotrichales) is reported as a lichen photobiont for the first time and is the first of its kind in the order. These results indicate that undescribed algal diversity is waiting to be uncovered in seashore lichens. [ABSTRACT FROM AUTHOR]

Ulva compressa, a green tide‐forming species, can adapt to hypo‐salinity conditions, such as estuaries and brackish lakes. To understand the underlying molecular mechanisms of hypo‐salinity stress tolerance, transcriptome‐wide gene expression profiles in U. compressa were created using digital gene expression profiles. The RNA‐seq data were analyzed based on the comparison of differently expressed genes involved in specific pathways under hypo‐salinity and recovery conditions. The up‐regulation of genes in photosynthesis and glycolysis pathways may contribute to the recovery of photosynthesis and energy metabolism, which could provide sufficient energy for the tolerance under long‐term hyposaline stress. Multiple strategies, such as ion transportation and osmolytes metabolism, were performed to maintain the osmotic homeostasis. Additionally, several long noncoding RNA were differently expressed during the stress, which could play important roles in the osmotolerance. Our work will serve as an essential foundation for the understanding of the tolerance mechanism of U. compressa under the fluctuating salinity conditions. [ABSTRACT FROM AUTHOR]

The primarily freshwater genus Chara is comprised of many species that exhibit a wide range of salinity tolerance. The range of salt tolerance provides a good platform for investigating the role of transport mechanisms in response to salt stress, and the close evolutionary relationship between Charophytes and land plants can provide broader insights. We investigated the response to salt stress of previously identified transport mechanisms in two species of Chara, Chara longifolia (salt‐tolerant), and Chara australis (salt‐sensitive): a cation transporter (HKT), a Na+/H+ antiport (NHX), H+‐ATPase (AHA), and a Na+‐ATPase (ENA). The presence of these candidate genes has been confirmed in both species of Chara, with the exception of the Na+‐ATPase, which is present only in salt‐tolerant Chara longifolia. Time‐course Illumina transcriptomes were created using RNA from multiple time points (0, 6, 12, 24 and 48 h) after freshwater cultures for each species were exposed to salt stress. These transcriptomes verified our hypotheses of these mechanisms conferring salt tolerance in the two species examined and also aided in identification of specific transcripts representing our genes of interest in both species. The expression of these transcripts was validated through use of qPCR, in a similar experimental set‐up used for the RNAseq data described above. The RNAseq and qPCR data showed significant changes of expression mechanisms in C. longifolia (respectively), a down‐regulation of HKT and a substantial up‐regulation of ENA. Significant responses to salt stress in salt‐sensitive C. australis show up‐regulation of NHX and AHA. [ABSTRACT FROM AUTHOR]

To better understand the effect of temperature on the growth and nitrate reductase activity (NRA) of Ulva prolifera and their relationships, the effects of five different temperatures (10, 15, 20, 25, and 30°C) were investigated in a laboratory setup. In this study, an optimization in vitro analysis method for Ulva prolifera NRA was developed. Under different treatments, the NRA, nitrate concentration, pH, the intracellular nitrate and nitrite concentrations, and the POC/PON were evaluated. The results of the in vitro analysis method showed it was optimal for the NRA assay when the extraction time was 6 min, enzymatic reaction time 30 min, volume of phenazine methosulfate (PMS) solution 50 μL, NADH concentration 0.36 mM, and KNO3 concentration 10 mM. The maximal NRA (NRAmax) appeared on the 2nd day in the 10, 15, and 20°C (low‐temperature) groups and on the 1st day in the 25 and 30°C (high‐temperature) groups. The algal growth ended earlier at a high temperature, ending after 5 d at 30 and 25°C and 7 d at 20°C and 9 d at 15°C, and the alga at 10°C had been growing during the incubation period. Ulva prolifera cultivated in a range of 10–20°C had a long growth cycle and the NRA decreased with increasing temperature when exceeded 15°C, a positive correlation between algal growth and NRA was observed. This study supports NRA is a suitable proxy of the effects of temperature changes on the ability of Ulva prolifera to uptake and metabolize nitrogen nutrients. [ABSTRACT FROM AUTHOR]

Volvox barberi is a multicellular green alga forming spherical colonies of 10,000–50,000 differentiated somatic and germ cells. We observed that in culture, these colonies actively self‐organized in just a few minutes into "flocks" that contained as many as 100 colonies moving and rotating collectively for hours. The colonies in flocks formed two‐dimensional, irregular, active crystals, that is, geometric lattices within which individual colonies rotated separately. These groupings sometimes disassembled back into individual colonies just as quickly, but in some cases, flocks persisted over several hours. Close inspection of flock formation in the presence of a tracer dye suggested that colony and flock rotations were producing vortices in the fluid medium over a range spanning multiple flock diameters, perhaps providing a physical mechanism for aggregation. [ABSTRACT FROM AUTHOR]

The gametes of chlorophytes can be divided into two morphological types (types α and β) based on the position of the mating structure relative to the flagella and eyespot. To elucidate the relationship between the morphological types and the sexes, we studied spatial relationships between the flagellar apparatus‐eyespot‐mating structures in biflagellate male and female gametes and their fate after fertilization in the anisogamous (Monostroma angicava) and the slightly anisogamous species (Collinsiella cava) using field emission scanning electron microscopy and transmission electron microscopy. The smaller male and larger female gametes of M. angicava had two basal bodies arranged at a 180° angle and the cell surface coated with square‐shaped body scales, except for the flagella and mating structures. The mating structure of the female gamete was located on the same side of the flagellar beat plane as the eyespot (type β), whereas that of the male gamete was located on the opposite side (type α). This mating structure arrangement was also confirmed in C. cava. The initial fusion when male and female gametes were mixed involved the mating structures. In a fusing pair of gametes, each flagellum of one gamete lay alongside one flagellum of the other gamete. As fusion proceeded, the gamete pair transformed into a quadriflagellate planozygote, in which the four basal bodies were arranged in a cruciate pattern. The eyespots were positioned side‐by‐side on the same side of the cell. These results suggest that the two morphological types of gametes are intimately correlated with the particular sexes. [ABSTRACT FROM AUTHOR]

Many organisms including unicellular (diatoms, radiolaria, and chrysophytes), higher plants (rice and horsetail) and animals (sponges) use silica as a main part of skeletons. The bioavailable form of silicon is silicic acid and the mechanism of silicic acid penetration into living cells is still an enigma. Macropinocytosis was assumed as a key stage of the silicon capture by diatoms but assimilation of monomeric silicic acid by this way requires enormous amounts of water to be passed through the cell. We hypothesized that silicon can be captured by diatoms via endocytosis in the form of partially condensed silicic acid (oligosilicates) whose formation on the diatom surface was supposed. Oligosilicates are negatively charged nanoparticles and similar to coils of poly(acrylic acid) (PAA). We have synthesized fluorescent tagged PAA as well as several neutral and positively charged polymers. Cultivation of the diatom Ulnaria ferefusiformis in the presence of these polymers showed that only PAA is able to penetrate into siliceous frustules. The presence of PAA in the frustules was confirmed with chromatography and PAA causes various aberrations of the valve morphology. Growth of U. ferefusiformis and two other diatoms in the presence of tri‐ and tetracarbonic fluorescent tagged acids points to the ability of diatoms to recognize substances that bear four acidic groups and to include them into siliceous frustules. Thus, partial condensation of silicic acid is a plausible first stage of silicon assimilation. [ABSTRACT FROM AUTHOR]

According the a strong tradition St. Thomas, a disciple of Jesus Christ arrived in Keralam in 52 A.D. and established the Church. AS this Church was guided in administration by the East Syrian Church of Persia, the St. Thomas Christians came to be known also as Syrian Christians. The first Christian community in Keralam seems to have assimilated the caste system of Keralam. They also claimed their descent from Namputris, the Brahmin caste of Keralam, although this claim is refuted. It is also said that the caste feelings made early Christians uninterested in proselytisation, and conversions from the lower castes began only in the 16th century. After conversion the Christians continued to be subjected to caste discrimination by the other Christians. The traditional Christians and the Church of Keralam seem to have failed the dalit Christians. The traditional Christians should shed their claim of having descended from upper caste and the consequent caste values. The Church should accept the dalits in the development process as equals. Above all the dalit Christians of Keralam should organise themselves, assert their identity and try to seek their development by themselves. [ABSTRACT FROM AUTHOR]

Nutrient deprivation induces reserve accumulation in unicellular algae. An absence of nitrogen in the growth media results in the reorganization of the photosynthetic apparatus and triggers an increase in starch and triacylglyceride (TAG) accumulation in different algal species. Here we study the integration of photosynthetic regulatory mechanisms with carbon partitioning under N stress in C. reinhardtii. The mutant, proton gradient regulation 5 (pgr5) is impaired in photosynthetic cyclic electron flow resulting in low chloroplastic ATP/NADPH ratios. Over a time course, under both mixotrophic and phototrophic conditions, the pgr5 mutant did not accumulate starch in the first three days, but rather degraded its meagre reserves. In contrast, there was a high TAG content in the pgr5 mutant which we show, is not linked to a selective increase in autophagy in pgr5. In all strains, proteins involved in alternative electron pathways are upregulated while Photosystem II and chlorophyll are strongly degraded; pgr5 only preferentially preserved some cyt b 6 f complex. Our results show that low ATP/NADPH ratios due to an absence of cyclic electron flow in pgr5 result in the mobilization of starch and strong TAG accumulation from the onset of N stress in Chlamydomonas., (© 2024 The Author(s). Physiologia Plantarum published by John Wiley & Sons Ltd on behalf of Scandinavian Plant Physiology Society.)

Using a mutant screen, we identified trehalose 6-phosphate phosphatase 1 (TSPP1) as a functional enzyme dephosphorylating trehalose 6-phosphate (Tre6P) to trehalose in Chlamydomonas reinhardtii. The tspp1 knock-out results in reprogramming of the cell metabolism via altered transcriptome. As a secondary effect, tspp1 also shows impairment in 1 O 2 -induced chloroplast retrograde signalling. From transcriptomic analysis and metabolite profiling, we conclude that accumulation or deficiency of certain metabolites directly affect 1 O 2 -signalling. 1 O 2 -inducible GLUTATHIONE PEROXIDASE 5 (GPX5) gene expression is suppressed by increased content of fumarate and 2-oxoglutarate, intermediates in the tricarboxylic acid cycle (TCA cycle) in mitochondria and dicarboxylate metabolism in the cytosol, but also myo-inositol, involved in inositol phosphate metabolism and phosphatidylinositol signalling system. Application of another TCA cycle intermediate, aconitate, recovers 1 O 2 -signalling and GPX5 expression in otherwise aconitate-deficient tspp1. Genes encoding known essential components of chloroplast-to-nucleus 1 O 2 -signalling, PSBP2, MBS, and SAK1, show decreased transcript levels in tspp1, which also can be rescued by exogenous application of aconitate. We demonstrate that chloroplast retrograde signalling involving 1 O 2 depends on mitochondrial and cytosolic processes and that the metabolic status of the cell determines the response to 1 O 2 ., (© 2023. The Author(s).)

Greenhouse gas (GHG) emissions have created a global climate crisis which requires immediate interventions to mitigate the negative effects on all aspects of life on this planet. As current agriculture and land use contributes up to 25% of total GHG emissions, plant scientists take center stage in finding possible solutions for a transition to sustainable agriculture and land use. In this article, the PlantACT! (Plants for climate ACTion!) initiative of plant scientists lays out a road map of how and in which areas plant scientists can contribute to finding immediate, mid-term, and long-term solutions, and what changes are necessary to implement these solutions at the personal, institutional, and funding levels., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

In land plants and cyanobacteria, co-translational association of chlorophyll (Chl) to the nascent D1 polypeptide, a reaction center protein of photosystem II (PSII), requires a Chl binding complex consisting of a short-chain dehydrogenase (high chlorophyll fluorescence 244 [HCF244]/uncharacterized protein 39 [Ycf39]) and one-helix proteins (OHP1 and OHP2 in chloroplasts) of the light-harvesting antenna complex superfamily. Here, we show that an ohp2 mutant of the green alga Chlamydomonas (Chlamydomonas reinhardtii) fails to accumulate core PSII subunits, in particular D1 (encoded by the psbA mRNA). Extragenic suppressors arose at high frequency, suggesting the existence of another route for Chl association to PSII. The ohp2 mutant was complemented by the Arabidopsis (Arabidopsis thaliana) ortholog. In contrast to land plants, where psbA translation is prevented in the absence of OHP2, ribosome profiling experiments showed that the Chlamydomonas mutant translates the psbA transcript over its full length. Pulse labeling suggested that D1 is degraded during or immediately after translation. The translation of other PSII subunits was affected by assembly-controlled translational regulation. Proteomics showed that HCF244, a translation factor which associates with and is stabilized by OHP2 in land plants, still partly accumulates in the Chlamydomonas ohp2 mutant, explaining the persistence of psbA translation. Several Chl biosynthesis enzymes overaccumulate in the mutant membranes. Partial inactivation of a D1-degrading protease restored a low level of PSII activity in an ohp2 background, but not photoautotrophy. Taken together, our data suggest that OHP2 is not required for psbA translation in Chlamydomonas, but is necessary for D1 stabilization., Competing Interests: Conflict of interest statement. None declared., (© American Society of Plant Biologists 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

A Chlamydomonas reinhardtii RuBisCO-less mutant, Δ rbcL , 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 Δ rbcL 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 Δ rbcL 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Saint-Sorny, Brzezowski, Arrivault, Alric and Johnson.)

Protein phosphorylation plays important roles in short-term regulation of photosynthetic electron transfer, and during state transitions, the kinase STATE TRANSITION7 (STT7) of Chlamydomonas reinhardtii phosphorylates components of light-harvesting antenna complex II (LHCII). This reversible phosphorylation governs the dynamic allocation of a part of LHCII to PSI or PSII, depending on light conditions and metabolic demands, but counteracting phosphatase(s) remain unknown in C. reinhardtii Here we analyzed state transitions in C. reinhardtii mutants of two phosphatases, PROTEIN PHOSPHATASE1 and PHOTOSYSTEM II PHOSPHATASE, which are homologous to proteins that antagonize the state transition kinases (STN7 and STN8) in Arabidopsis ( Arabidopsis thaliana ). The transition from state 2 to state 1 was retarded in pph1 , and surprisingly also in pbcp However, both mutants eventually returned to state 1. In contrast, the double mutant pph1 ; pbcp appeared strongly locked in state 2. The complex phosphorylation patterns of the LHCII trimers and of the monomeric subunits were affected in the phosphatase mutants. Their analysis indicated that the two phosphatases have different yet overlapping sets of protein targets. The dual control of thylakoid protein dephosphorylation and the more complex antenna phosphorylation patterns in C. reinhardtii compared to Arabidopsis are discussed in the context of the stronger amplitude of state transitions and the more diverse LHCII isoforms in the alga., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

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 b 6 f 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., Competing Interests: The authors declare no competing interests.

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 b 6 f complex (Cytb 6 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 Cytb 6 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 Cytb 6 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 Cytb 6 f., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)

Photosynthetic organisms have evolved numerous photoprotective mechanisms and alternative electron sinks/pathways to fine-tune the photosynthetic apparatus under dynamic environmental conditions, such as varying carbon supply or fluctuations in light intensity. In cyanobacteria flavodiiron proteins (FDPs) protect the photosynthetic apparatus from photodamage under fluctuating light (FL). In Arabidopsis thaliana, which does not possess FDPs, the PGR5-related pathway enables FL photoprotection. The direct comparison of the pgr5, pgrl1 and flv knockout mutants of Chlamydomonas reinhardtii grown under ambient air demonstrates that all three proteins contribute to the survival of cells under FL, but to varying extents. The FDPs are crucial in providing a rapid electron sink, with flv mutant lines unable to survive even mild FL conditions. In contrast, the PGRL1 and PGR5-related pathways operate over relatively slower and longer time-scales. Whilst deletion of PGR5 inhibits growth under mild FL, the pgrl1 mutant line is only impacted under severe FL conditions. This suggests distinct roles, yet a close relationship, between the function of PGR5, PGRL1 and FDP proteins in photoprotection., (© 2018 The Authors. The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.)

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 b 6 f 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., (© 2018 American Society of Plant Biologists. All rights reserved.)

The cytochrome (cyt) b 6 f 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 b 6 f 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 b 6 f complex, and complementation of a Δ petD 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 b 6 f subunit IV are crucial for state transitions. In vitro reconstitution experiments with purified cyt b 6 f and recombinant Stt7 kinase domain show that cyt b 6 f enhances Stt7 autophosphorylation and that the Arg125 residue is directly involved in this process. The peripheral stromal structure of the cyt b 6 f complex had, until now, no reported function. Evidence is now provided of a direct interaction with Stt7 on the stromal side of the membrane., Competing Interests: The authors declare no conflict of interest., (Published under the PNAS license.)

Photosynthetic reactions proceed along a linear electron transfer chain linking water oxidation at photosystem II (PSII) to CO 2 reduction in the Calvin-Benson-Bassham cycle. Alternative pathways poise the electron carriers along the chain in response to changing light, temperature and CO 2 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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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.

The nucleo-cytoplasmic compartment exerts anterograde control on chloroplast gene expression through numerous proteins that intervene at posttranscriptional steps. Here, we show that the maturation of psaC mutant (mac1) of Chlamydomonas reinhardtii is defective in photosystem I and fails to accumulate psaC mRNA. The MAC1 locus encodes a member of the Half-A-Tetratricopeptide (HAT) family of super-helical repeat proteins, some of which are involved in RNA transactions. The Mac1 protein localizes to the chloroplast in the soluble fraction. MAC1 acts through the 5' untranslated region of psaC transcripts and is required for their stability. Small RNAs that map to the 5'end of psaC RNA in the wild type but not in the mac1 mutant are inferred to represent footprints of MAC1-dependent protein binding, and Mac1 expressed in bacteria binds RNA in vitro. A coordinate response to iron deficiency, which leads to dismantling of the photosynthetic electron transfer chain and in particular of photosystem I, also causes a decrease of Mac1. Overexpression of Mac1 leads to a parallel increase in psaC mRNA but not in PsaC protein, suggesting that Mac1 may be limiting for psaC mRNA accumulation but that other processes regulate protein accumulation. Furthermore, Mac 1 is differentially phosphorylated in response to iron availability and to conditions that alter the redox balance of the electron transfer chain., (© 2016 American Society of Plant Biologists. All rights reserved.)

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.

Continuous hydrogen photo-production under sulfur deprivation was studied in the Chlamydomonas reinhardtii pgr5 pgrl1 double mutant and respective single mutants. Under medium light conditions, the pgr5 exhibited the highest performance and produced about eight times more hydrogen than the wild type, making pgr5 one of the most efficient hydrogen producer reported so far. The pgr5 pgrl1 double mutant showed an increased hydrogen burst at the beginning of sulfur deprivation under high light conditions, but in this case the overall amount of hydrogen produced by pgr5 pgrl1 as well as pgr5 was diminished due to photo-inhibition and increased degradation of PSI. In contrast, the pgrl1 was effective in hydrogen production in both high and low light. Blocking photosynthetic electron transfer by DCMU stopped hydrogen production almost completely in the mutant strains, indicating that the main pathway of electrons toward enhanced hydrogen production is via linear electron transport. Indeed, PSII remained more active and stable in the pgr mutant strains as compared to the wild type. Since transition to anaerobiosis was faster and could be maintained due to an increased oxygen consumption capacity, this likely preserves PSII from photo-oxidative damage in the pgr mutants. Hence, we conclude that increased hydrogen production under sulfur deprivation in the pgr5 and pgrl1 mutants is caused by an increased stability of PSII permitting sustainable light-driven hydrogen production in Chlamydomonas reinhardtii.

Cyclic electron flow (CEF) around PSI regulates acceptor-side limitations and has multiple functions in the green alga, Chlamydomonas reinhardtii. Here we draw on recent and historic literature and concentrate on its role in Photosystem I (PSI) photoprotection, outlining causes and consequences of damage to PSI and CEF's role as an avoidance mechanism. We outline two functions of CEF in PSI photoprotection that are both linked to luminal acidification: firstly, its action on Photosystem II with non-photochemical quenching and photosynthetic control and secondly, its action in poising the stroma to overcome acceptor-side limitation by rebalancing NADPH and ATP ratios for carbon fixation.

In plants and algae, chloroplast gene expression is controlled by nucleus-encoded proteins that bind to mRNAs in a specific manner, stabilizing mRNAs or promoting their splicing, editing, or translation. Here, we present the characterization of two mRNA stabilization factors of the green alga Chlamydomonas reinhardtii, which both belong to the OctotricoPeptide Repeat (OPR) family. MCG1 is necessary to stabilize the petG mRNA, encoding a small subunit of the cytochrome b6 f complex, while MBI1 stabilizes the psbI mRNA, coding for a small subunit of photosystem II. In the mcg1 mutant, the small RNA footprint corresponding to the 5'-end of the petG transcript is reduced in abundance. In both cases, the absence of the small subunit perturbs assembly of the cognate complex. Whereas PetG is essential for formation of a functional cytochrome b6 f dimer, PsbI appears partly dispensable as a low level of PSII activity can still be measured in its absence. Thus, nuclear control of chloroplast gene expression is not only exerted on the major core subunits of the complexes, but also on small subunits with a single transmembrane helix. While OPR proteins have thus far been involved in translation or trans-splicing of plastid mRNAs, our results expand the potential roles of this repeat family to their stabilization., (© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.)

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., (© 2014 American Society of Plant Biologists. All rights reserved.)

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.

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.

Spectroscopic studies on photosynthetic electron transfer generally are based upon the monitoring of dark to light changes in the electron transfer chain. These studies, which focus on the light reactions of photosynthesis, also indirectly provide information on the redox or metabolic state of the chloroplast in the dark. Here, using the unicellular microalga Chlamydomonas reinhardtii, we study the impact of heterotrophic/mixotrophic acetate feeding on chloroplast carbon metabolism by using the spectrophotometric detection of P700(+), the photooxidized primary electron donor of photosystem I. We show that, when photosynthetic linear and cyclic electron flows are blocked (DCMU inhibiting PSII and methylviologen accepting electrons from PSI), the post-illumination reduction kinetics of P700(+) directly reflect the dark metabolic production of reductants (mainly NAD(P)H) in the stroma of chloroplasts. Such results can be correlated to other metabolic studies: in the absence of acetate, for example, the P700(+) reduction rate matches the rate of starch breakdown reported previously, confirming the chloroplast localization of the upstream steps of the glycolytic pathway in Chlamydomonas. Furthermore, the question of the interplay between photosynthetic and non-photosynthetic carbon metabolism can be addressed. We show that cyclic electron flow around photosystem I is twice as fast in a starchless mutant fed with acetate than it is in the WT, and we relate how changes in the flux of electrons from carbohydrate metabolism modulate the redox poise of the plastoquinone pool in the dark through chlororespiration.

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 b(6)f complex and PTOX2 were used to measure the oxidation rates of plastoquinols via PTOX1 and PTOX2. Those lacking both the cytochrome b(6)f complex and PTOX2 were more sensitive to light than the single mutants lacking either the cytochrome b(6)f 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.

The nuclear factor, Maturation/stability of RbcL (MRL1), regulates the accumulation of the chloroplast rbcL gene transcript in Chlamydomonas reinhardtii by stabilising the mRNA via its 5' UTR. An absence of MRL1 in algal mrl1 mutants leads to a complete absence of RuBisCO large subunit protein and thus a lack of accumulation of the RuBisCO holoenzyme. By complementing mrl1 mutants by random transformation of the nuclear genome with the MRL1 cDNA, different levels of rbcL transcript accumulate. We also observe that RuBisCO Large Subunit accumulation is perturbed. Complemented strains accumulating as little as 15% RuBisCO protein can grow phototrophically while RuBisCO in this range is limiting for phototrophic growth. We also observe that photosynthetic activity, here measured by the quantum yield of PSII, appears to be a determinant for phototrophic growth. In some strains that accumulate less RuBisCO, a strong production of reactive oxygen species is detected. In the absence of RuBisCO, oxygen possibly acts as the PSI terminal electron acceptor. These results show that random transformation of MRL1 into mrl1 mutants can change RuBisCO accumulation allowing a range of phototrophic growth phenotypes. Furthermore, this technique allows for the isolation of strains with low RuBisCO, within the range of acceptable photosynthetic growth and reasonably low ROS production. MRL1 is thus a potential tool for applications to divert electrons away from photosynthetic carbon metabolism towards alternative pathways., (© Springer Science+Business Media B.V. 2011)

Chlamydomonas reinhardtii, a unicellular green alga, has been exploited as a reference organism for identifying proteins and activities associated with the photosynthetic apparatus and the functioning of chloroplasts. Recently, the full genome sequence of Chlamydomonas was generated and a set of gene models, representing all genes on the genome, was developed. Using these gene models, and gene models developed for the genomes of other organisms, a phylogenomic, comparative analysis was performed to identify proteins encoded on the Chlamydomonas genome which were likely involved in chloroplast functions (or specifically associated with the green algal lineage); this set of proteins has been designated the GreenCut. Further analyses of those GreenCut proteins with uncharacterized functions and the generation of mutant strains aberrant for these proteins are beginning to unmask new layers of functionality/regulation that are integrated into the workings of the photosynthetic apparatus.

We identify and functionally characterize MRL1, a conserved nuclear-encoded regulator of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. The nonphotosynthetic mrl1 mutant of Chlamydomonas reinhardtii lacks ribulose-1,5-bisphosphate carboxylase/oxygenase, and the resulting block in electron transfer is partially compensated by redirecting electrons toward molecular oxygen via the Mehler reaction. This allows continued electron flow and constitutive nonphotochemical quenching, enhancing cell survival during illumination in spite of photosystem II and photosystem I photoinhibition. The mrl1 mutant transcribes rbcL normally, but the mRNA is unstable. The molecular target of MRL1 is the 5 ' untranslated region of rbcL. MRL1 is located in the chloroplast stroma, in a high molecular mass complex. Treatment with RNase or deletion of the rbcL gene induces a shift of the complex toward lower molecular mass fractions. MRL1 is well conserved throughout the green lineage, much more so than the 10 other pentatricopeptide repeat proteins found in Chlamydomonas. Depending upon the organism, MRL1 contains 11 to 14 pentatricopeptide repeats followed by a novel MRL1-C domain. In Arabidopsis thaliana, MRL1 also acts on rbcL and is necessary for the production/stabilization of the processed transcript, presumably because it acts as a barrier to 5 ' >3 ' degradation. The Arabidopsis mrl1 mutant retains normal levels of the primary transcript and full photosynthetic capacity.

Here, we describe a new imaging setup able to assess in vivo photosynthetic activity. The system specifically measures time-resolved chlorophyll fluorescence in response to light. It is composed of a fast digital camera equipped with a wide-angle lens for the analysis of samples up to 10 x 10 cm, i.e. entire plants or petri dishes. In the choice of CCD, we have opted for a 12-bits high frame rate [150 fps (frames per second)] at the expense of definition (640 x 480 pixels). Although the choice of digital camera is always a compromise between these two related features, we have designed a flexible system allowing the fast sampling of images (down to 100 micros) with a maximum spatial resolution. This image readout system, synchronized with actinic light and saturating pulses, allows a precise determination of F(0) and F(M), which is required to monitor PSII activity. This new imaging system, together with image processing techniques, is useful to investigate the heterogeneity of photosynthetic activity within leaves or to screen large numbers of unicellular algal mutant colonies to identify those with subtle changes in photosynthetic electron flow.

We have investigated the photosynthetic properties of Acaryochloris marina, a cyanobacterium distinguished by having a high level of chlorophyll d, which has its absorption bands shifted to the red when compared with chlorophyll a. Despite this unusual pigment content, the overall rate and thermodynamics of the photosynthetic electron flow are similar to those of chlorophyll a-containing species. The midpoint potential of both cytochrome f and the primary electron donor of photosystem I (P(740)) were found to be unchanged with respect to those prevailing in organisms having chlorophyll a, being 345 and 425 mV, respectively. Thus, contrary to previous reports (Hu, Q., Miyashita, H., Iwasaki, I. I., Kurano, N., Miyachi, S., Iwaki, M., and Itoh, S. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 13319-13323), the midpoint potential of the electron donor P(740) has not been tuned to compensate for the decrease in excitonic energy in A. marina and to maintain the reducing power of photosystem I. We argue that this is a weaker constraint on the engineering of the oxygenic photosynthetic electron transfer chain than preserving the driving force for plastoquinol oxidation by P(740), via the cytochrome b(6)f complex. We further show that there is no restriction in the diffusion of the soluble electron carrier between cytochrome b(6)f and photosystem I in A. marina, at variance with plants. This difference probably reflects the simplified ultrastructure of the thylakoids of this organism, where no segregation into grana and stroma lamellae is observed. Nevertheless, chlorophyll fluorescence measurements suggest that there is energy transfer between adjacent photosystem II complexes but not from photosystem II to photosystem I, indicating spatial separation between the two photosystems.

Physiological and genetic studies with the ramosus (rms) mutants in garden pea (Pisum sativum) and more axillary shoots (max) mutants in Arabidopsis (Arabidopsis thaliana) have shown that shoot branching is regulated by a network of long-distance signals. Orthologous genes RMS1 and MAX4 control the synthesis of a novel graft-transmissible branching signal that may be a carotenoid derivative and acts as a branching inhibitor. In this study, we demonstrate further conservation of the branching control system by showing that MAX2 and MAX3 are orthologous to RMS4 and RMS5, respectively. This is consistent with the long-standing hypothesis that branching in pea is regulated by a novel long-distance signal produced by RMS1 and RMS5 and that RMS4 is implicated in the response to this signal. We examine RMS5 expression and show that it is more highly expressed relative to RMS1, but under similar transcriptional regulation as RMS1. Further expression studies support the hypothesis that RMS4 functions in shoot and rootstock and participates in the feedback regulation of RMS1 and RMS5 expression. This feedback involves a second novel long-distance signal that is lacking in rms2 mutants. RMS1 and RMS5 are also independently regulated by indole-3-acetic acid. RMS1, rather than RMS5, appears to be a key regulator of the branching inhibitor. This study presents new interactions between RMS genes and provides further evidence toward the ongoing elucidation of a model of axillary bud outgrowth in pea.

A sucrose: sucrose 1-fructosyltransferase (1-SST) gene and cDNA (Lp 1-SST) from perennial ryegrass (Lolium perenne) were isolated. The Lp 1-SST gene was fully sequenced and shown to contain three exons and two introns. Nucleotide sequence analysis of the 4824 bp Lp 1-SST genomic sequence revealed 1618 bp of 5' UTR and an open reading frame of 1962 bp encoding a protein of 653 amino acids. Lp 1-SST is 95% identical to the tall fescue 1-SST and contains plant fructosyltransferase functional domains. Lp 1-SST corresponds to a single copy gene in perennial ryegrass, and is expressed in young leaf bases and mature leaf sheaths. The recombinant Lp 1-SST protein from corresponding cDNA expression in Pichia pastoris showed 1-SST activity.

An invertase (LpFT2) cDNA from perennial ryegrass was isolated and sequenced. Nucleotide sequence analysis revealed an ORF of 2016 bp encoding a protein of 671 amino acids. LpFT2 is 76% identical to sugarcane soluble acid invertase, and contains invertase and fructosyltransferase functional domains. LpFT2 is present as a single copy gene and maps to the distal region of LG6 in perennial ryegrass. The expression pattern analysis of LpFT2 revealed transcript accumulation in seedlings and in mature leaf sheaths. The LpFT2 recombinant protein expressed in yeast showed invertase and fructan exohydrolase-like activities with complete breakdown of sucrose, 1-kestose (DP3), 1,1-kestotetraose (DP4) and 1,1,1-kestopentaose (DP5) into glucose and fructose.