Photosynthetic Model Organism Research Articles

Subcellular proteomics for determining iron-limited remodeling of plastids in the model diatom Thalassiosira pseudonana (Bacillariophyta).

Diatoms are important primary producers in the world's oceans, yet their growth is constrained in large regions by low bioavailable iron (Fe). Low-Fe stress-induced limitation of primary production is due to requirements for Fe in components of essential metabolic pathways including photosynthesis and other chloroplast plastid functions. Studies have shown that under low-Fe stress, diatoms alter plastid-specific processes, including components of electron transport. These physiological changes suggest changes of protein content and in protein abundances within the diatom plastid. While in silico predictions provide putative information on plastid-localized proteins, knowledge of diatom plastid proteins remains limited in comparison to well-studied model photosynthetic organisms. To address this, we employed shotgun proteomics to investigate the proteome of subcellular plastid-enriched fractions from Thalassiosira pseudonana to gain a better understanding of how the plastid proteome is remodeled in response to Fe limitation. Using mass spectrometry-based peptide identification and quantification, we analyzed T. pseudonana grown under Fe-replete and -limiting conditions. Through these analyses, we inferred the relative quantities of each protein, revealing that Fe limitation regulates major metabolic pathways in the plastid, including the Calvin cycle. Additionally, we observed changes in the expression of light-harvesting proteins. In silico localization predictions of proteins identified in this plastid-enriched proteome allowed for an in-depth comparison of theoretical versus observed plastid-localization, providing evidence for the potential of additional protein import pathways into the diatom plastid.

Circadian regulation of metabolism across photosynthetic organisms.

Circadian regulation produces a biological measure of time within cells. The daily cycle in the availability of light for photosynthesis causes dramatic changes in biochemical processes in photosynthetic organisms, with the circadian clock having crucial roles in adaptation to these fluctuating conditions. Correct alignment between the circadian clock and environmental day-night cycles maximizes plant productivity through its regulation of metabolism. Therefore, the processes that integrate circadian regulation with metabolism are key to understanding how the circadian clock contributes to plant productivity. This forms an important part of exploiting knowledge of circadian regulation to enhance sustainable crop production. Here, we examine the roles of circadian regulation in metabolic processes in source and sink organ structures of Arabidopsis. We also evaluate possible roles for circadian regulation in root exudation processes that deposit carbon into the soil, and the nature of the rhythmic interactions between plants and their associated microbial communities. Finally, we examine shared and differing aspects of the circadian regulation of metabolism between Arabidopsis and other model photosynthetic organisms, and between circadian control of metabolism in photosynthetic and non-photosynthetic organisms. This synthesis identifies a variety of future research topics, including a focus on metabolic processes that underlie biotic interactions within ecosystems.

Enhancing overall carbon fixation and energy conversion with formate in green microalga Chlamydomonas reinhardtii

The natural carbon-concentration mechanism (CCM) is the solution to achieve high photosynthetic efficiency. Mimicking the natural CCM, exogenous formate which can be produced by renewable energy and CO2 artificially, was introduced into a model photosynthetic organism, Chlamydomonas reinhardtii, to improve the overall photosynthesis. To release the adverse effects of formate, the combination of different strategies, including co-substrate, evolution, and conditions optimization, the formate tolerance to over 40 mM, which was higher than previous irreversible inhibition limitation of formate on C. reinhardtii, was achieved, and formate-mediated enhancement of growth and photosynthesis activity was obtained with a 27 % biomass improvement and a 60 % increase in photosynthetic oxygen evolution. Interestingly, 82 % of formate was converted with the illumination, indicating the existence of energy interaction between photosynthesis and formate assimilation. Meanwhile, formate suppressed respiration and reduced a 29 % biomass decline in the night. Moreover, supported by the enzyme inhibitor, formate consumption, and biochemical compositions, it was postulated that formate was assimilated via the pyruvate formate-lyase (PFL) enzyme to stimulate the increase of photosynthetic components to maintain photosynthesis activity. With the overexpressed PFL and conditions optimization, formate could be further used as the sole organic carbon source for higher biomass in C. reinhardtii. This work presents the potentiality of introducing formate as artificial carbon intermediates to natural metabolic pathways for higher-than-nature photosynthetic efficiency.

Effect of Common ER Stress-Inducing Drugs on the Growth and Lipid Phenotypes of Chlamydomonas and Arabidopsis.

Endoplasmic reticulum (ER) stress is caused by the stress-induced accumulation of unfolded proteins in the ER. Several compounds are used to induce the unfolded protein response (UPR) in animals, with different modes of action, but which ER stress-inducing drugs induce ER stress in microalgae or land plants is unclear. In this study, we examined the effects of seven chemicals that were reported to induce ER stress in animals on the growth, UPR gene expression and fatty acid profiles of Chlamydomonas reinhardtii (Chlamydomonas) and Arabidopsis thaliana (Arabidopsis): 2-deoxyglucose, dithiothreitol (DTT), tunicamycin (TM), thapsigargin, brefeldin A (BFA), monensin (MON) and eeyarestatin I. In both model photosynthetic organisms, DTT, TM, BFA and MON treatment induced ER stress, as indicated by the induction of spliced bZIP1 and bZIP60, respectively. In Chlamydomonas, DTT, TM and BFA treatment induced the production of transcripts related to lipid biosynthesis, but MON treatment did not. In Arabidopsis, DTT, TM, BFA and MON inhibited seed germination and seedling growth with the activation of bZIP60. These findings lay the foundation for using four types of ER stress-inducing drugs in photosynthetic organisms, and they help uncover the mode of action of each compound.

Biochemistry and Biotechnology of Lipid Accumulation in the Microalga Nannochloropsis oceanica.

Oils are among the most important agricultural commodities and have wide applications in food/nutrition, biofuels, and oleochemicals. The oleaginous microalga Nannochloropsis oceanica can produce large amounts of oils and the high-value ω-3 eicosapentaenoic acid, which represents a promising resource for oil production targeting biodiesel, nutraceutical, and aquaculture industries. In recent years, with the availability of omics databases and the development of genetic tools, N. oceanica has been extensively investigated as a model photosynthetic organism for studying lipid metabolism and as a green cellular factory to produce lipids for industrial applications. This review summarizes the current knowledge on the lipid composition and biosynthetic pathways of N. oceanica and reviews the recent advances in metabolic engineering of lipid production in this microalga.

Impact of salinities, metals and organic compounds found in saline oil & gas produced water on microalgae and cyanobacteria

This work evaluates the impact of salinity and the toxicity of some metals and organic compounds commonly found in produced waters on the growth of model photosynthetic organisms. Five strains of marine microalgae and one cyanobacteria (i.e. Dunaliella salina, Nannochloropsis oceanica, Tetraselmis suecica, Picochlorum costavermella, Coccomyxa simplex and Synechococcus rubescens) were tested in microplates as well as the freshwater Chlorella vulgaris selected as reference. Results revealed that D.salina was able to growth at high salinity (up to 135 g·L−1). Copper was the most toxic metal for all strains (half maximal effective concentration between 0.1 and 10 mg·L−1) except for D.salina and C.simplex. These two strains were the most resistant to all metals tested. All organic compounds presented half maximal effective concentration above 10 mg·L−1, none of them being very toxic for the studied microorganisms. P.costavermella and C.simplex were the most resistant strains to organic compounds. Looking at tolerance to salinity, metals and organic compounds, D.salina appeared to be the best choice for biomass production in produced waters. In addition, growths in 80% artificial produced water supplemented with f medium confirm the feasibility to use this medium to produce biomass.

Comparative transcriptome analysis of short-term responses to salt and glycerol hyperosmotic stress in the green alga Dunaliella salina

Dunaliella salina is a model photosynthetic organism for studying osmoregulation. To further understand the molecular basis of short-term hyperosmotic-stress responses, gene expression was investigated by RNA-Seq of D. salina cells sampled at 15, 30, 60, and 120 min following exposure to salt and glycerol stress. De novo reconstruction of the transcriptome using Illumina paired-end reads generated 155,996 contigs, out of which 31,302 transcripts could be annotated by gene ontology, assigning them to 64 functional groups. Further functional analysis of the transcripts was performed using KEGG Enzyme Commission numbers. Out of a total of 4415 transcripts with more than two-fold differential expression, 479 transcripts were identified representing 414 enzyme commission numbers that were mapped to 120 KEGG pathways. The “Chromosome and associated proteins”, “Transporters”, “Cytoskeleton proteins”, and 92 other KEGG pathways mapped for differentially expressed transcripts were common to salt stress and glycerol stress. In addition, differentially expressed transcripts mapped uniquely to 22 metabolic pathways for glycerol stress and 7 metabolic pathways mapped uniquely for salt stress. The transcripts mapping to core carbon metabolism within starch, glycolysis, Calvin cycle, and glycerol metabolic pathways were analyzed and 19 transcripts showed differential expression. Cluster analysis resulted in the identification of 939 transcripts, which showed similar expression trends. Among the fastest and most strongly induced genes, glyoxylate cycle and fatty acid desaturation-related genes implied the induction of photorespiration following rapid shifts in the osmolarity of the growth medium. These results provide novel insights for further analyses of physiological responses to short-term osmotic shock.

Alcohol stress on cyanobacterial membranes: New insights revealed by transcriptomics.

Cyanobacteria are model photosynthetic prokaryotic organisms often used in biotechnology to produce biofuels including alcohols. The effect of alcohols on cyanobacterial cell physiology and specifically on membrane fluidity is poorly understood. Previous research on various primary aliphatic alcohols found that alcohols with a short hydrocarbon chain (C1-C3) do not affect expression of genes related to membrane physical state. In addition, less water-soluble alcohols with a hydrocarbon chain longer than C8 are found to have a reduced ability to reach cellular membranes hence do not drastically change membrane physical state or induce expression of stress-responsive genes. Therefore, hexan-1-ol (C6) is suggested to have the most profound effect on cyanobacterial membrane physical state. Here, we studied the effects of hexan-1-ol on the cyanobacterium Synechocystis sp. PCC 6803 transcriptome. The transcriptome data obtained is compared to the previously reported analysis of gene expression induced by benzyl alcohol and butan-1-ol. The set of genes whose expression is induced after exposure to all three studied alcohols is identified. The expression under alcohol stress for several general stress response operons is analyzed, and examples of antisense interactions of RNA are investigated.

Label-Free Quantitative Phosphoproteomics for Algae.

The unicellular alga Chlamydomonas reinhardtii is a model photosynthetic organism for the study of microalgal processes. Along with genomic and transcriptomic studies, proteomic analysis of Chlamydomonas has led to an increased understanding of its metabolic signaling as well as a growing interest in the elucidation of its phosphorylation networks. To this end, mass spectrometry-based proteomics has made great strides in large-scale protein quantitation as well as analysis of posttranslational modifications (PTMs) in a high-throughput manner. An accurate quantification of dynamic PTMs, such as phosphorylation, requires high reproducibility and sensitivity due to the substoichiometric levels of modified peptides, which can make depth of coverage challenging. Here we present a method using TiO2-based phosphopeptide enrichment paired with label-free LC-MS/MS for phosphoproteome quantification. Three technical replicate samples in Chlamydomonas were processed and analyzed using this approach, quantifying a total of 1775 phosphoproteins with a total of 3595 phosphosites. With a median CV of 21% across quantified phosphopeptides, implementation of this method for differential studies provides highly reproducible analysis of phosphorylation events. While the culturing and extraction methods used are specific to facilitate coverage in algal species, this approach is widely applicable and can easily extend beyond algae to other photosynthetic organisms with minor modifications.

The endogenous redox rhythm is controlled by a central circadian oscillator in cyanobacterium Synechococcus elongatus PCC7942.

The intracellular redox and the circadian clock in photosynthetic organisms are two major regulators globally affecting various biological functions. Both of the global control systems have evolved as systems to adapt to regularly or irregularly changing light environments. Here, we report that the two global regulators mutually interact in cyanobacterium Synechococcus elongatus PCC7942, a model photosynthetic organism whose clock molecular mechanism is well known. Electrochemical assay using a transmembrane electron mediator revealed that intracellular redox of S. elongatus PCC7942 cell exhibited circadian rhythms under constant light conditions. The redox rhythm disappeared when transcription/translation of clock genes is defunctionalized, indicating that the transcription/translation controlled by a core KaiABC oscillator generates the circadian redox rhythm. Importantly, the amplitude of the redox rhythm at a constant light condition was large enough to affect the KaiABC oscillator. The findings indicated that the intracellular redox state is actively controlled to change in a 24-h cycle under constant light conditions by the circadian clock system.

Characterization of the GPR1/FUN34/YaaH protein family in the green microalga Chlamydomonas suggests their role as intracellular membrane acetate channels.

The unicellular green microalga Chlamydomonas reinhardtii is a powerful photosynthetic model organism which is capable of heterotrophic growth on acetate as a sole carbon source. This capacity has enabled its use for investigations of perturbations in photosynthetic machinery as mutants can be recovered heterotrophically. Fixation of acetate into cellular carbon metabolism occurs first by its conversion into acetyl‐CoA by a respective synthase and the generation of succinate by the glyoxylate cycle. These metabolic steps have been recently determined to largely occur in the peroxisomes of this alga; however, little is known about the trafficking and import of acetate or its subcellular compartmentalization. Recently, the genes of five proteins belonging to the GPR1/FUN34/YaaH (GFY) superfamily were observed to exhibit increased expression in C. reinhardtii upon acetate addition, however, no further characterization has been reported. Here, we provide several lines of evidence to implicate Cr GFY1–5 as channels which share structural homology with bacterial succinate‐acetate channels and specifically localize to microbodies, which are surprisingly distinct from the glyoxylate cycle‐containing peroxisomes. We demonstrate structural models, gene expression profiling, and in vivo fluorescence localization of all five isoforms in the algal cell to further support this role.

Singlet oxygen imaging using fluorescent probe Singlet Oxygen Sensor Green in photosynthetic organisms

Formation of singlet oxygen (1O2) was reported to accompany light stress in plants, contributing to cell signaling or oxidative damage. So far, Singlet Oxygen Sensor Green (SOSG) has been the only commercialized fluorescent probe for 1O2 imaging though it suffers from several limitations (unequal penetration and photosensitization) that need to be carefully considered to avoid misinterpretation of the analysed data. Herein, we present results of a comprehensive study focused on the appropriateness of SOSG for 1O2 imaging in three model photosynthetic organisms, unicellular cyanobacteria Synechocystis sp. PCC 6803, unicellular green alga Chlamydomonas reinhardtii and higher plant Arabidopsis thaliana. Penetration of SOSG differs in both unicellular organisms; while it is rather convenient for Chlamydomonas it is restricted by the presence of mucoid sheath of Synechocystis, which penetrability might be improved by mild heating. In Arabidopsis, SOSG penetration is limited due to tissue complexity which can be increased by pressure infiltration using a shut syringe. Photosensitization of SOSG and SOSG endoperoxide formed by its interaction with 1O2 might be prevented by illumination of samples by a red light. When measured under controlled conditions given above, SOSG might serve as specific probe for detection of intracellular 1O2 formation in photosynthetic organisms.

Highly Time-Resolved Metabolic Reprogramming toward Differential Levels of Phosphate in Chlamydomonas reinhardtii.

Understanding phosphorus metabolism in photosynthetic organisms is important as it is closely associated with enhanced crop productivity and pollution management for natural ecosystems (e.g., algal blooming). Accordingly, we exploited highly time-resolved metabolic responses to different levels of phosphate deprivation in Chlamydomonas reinhardtii, a photosynthetic model organism. We conducted non-targeted primary metabolite profiling using gas-chromatography time-of-flight mass spectrometric analysis. Primarily, we systematically identified main contributors to degree-wise responses corresponding to the levels of phosphate deprivation. Additionally, we systematically characterized the metabolite sets specific to different phosphate conditions and their interactions with culture time. Among them were various types of fatty acids that were most dynamically modulated by the phosphate availability and culture time in addition to phosphorylated compounds.

Probing the Global Kinome and Phosphoproteome in Chlamydomonas reinhardtii via Sequential Enrichment and Quantitative Proteomics

Identifying dynamic protein phosphorylation events is critical for understanding kinase/phosphatase regulated signaling pathways. Research aimed at dissecting the roles of kinases on a global “‐omics” scale has grown within the past decade with the potential to become more comprehensive for discovery‐based, untargeted kinome analyses. Utilization of immobilized kinase inhibitors [e.g., multiplexed inhibitor beads (MIBs) or kinobeads] broadly captures protein kinases through binding to the ATP‐binding domain of intact native kinases. To date, protein phosphorylation and kinase expression have been examined independently in photosynthetic organisms. Presented here is a method to study the global kinome and phosphoproteome in tandem in the model photosynthetic organism, the alga Chlamydomonas reinhardtii (Chlamydomonas), using mass spectrometry‐based label‐free proteomics. A dual enrichment strategy targets intact protein kinases via capture on MIBs columns with subsequent proteolytic digestion of unbound proteins and peptide‐based phosphorylation enrichment. To increase depth of coverage, both data‐dependent and data‐independent (via SWATH) mass spectrometric acquisitions were performed to obtain a >50% increase in coverage of the enriched Chlamydomonas kinome over coverage found with no enrichment. The quantitative kinome and phosphoproteomic datasets yield 115 protein kinases and 2,250 phosphopeptides with 1,314 localized phosphosites with excellent reproducibility across biological replicates (90% of quantified sites with coefficient of variation below 11%). This approach enables simultaneous investigation of kinases and phosphorylation events at the global level to facilitate understanding of kinase networks and their influence in cell signaling events. For example, through the induction of stress in Chlamydomonas and the use of differential proteomic studies of the kinome and phosphoproteome, future studies using this tandem enrichment have the potential to target specific pathways and investigate global kinome and phosphoproteome changes in a quantitative fashion.Support or Funding InformationThis research was supported by a National Science Foundation CAREER award (MCB‐1552522) to L.M.H. Authors would like to acknowledge Dr. Gary Johnson for providing the MIBs beads.

Probing the global kinome and phosphoproteome in Chlamydomonas reinhardtii via sequential enrichment and quantitative proteomics.

The identification of dynamic protein phosphorylation events is critical for understanding kinase/phosphatase-regulated signaling pathways. To date, protein phosphorylation and kinase expression have been examined independently in photosynthetic organisms. Here we present a method to study the global kinome and phosphoproteome in tandem in a model photosynthetic organism, the alga Chlamydomonas reinhardtii (Chlamydomonas), using mass spectrometry-based label-free proteomics. A dual enrichment strategy targets intact protein kinases via capture on immobilized multiplexed inhibitor beads with subsequent proteolytic digestion of unbound proteins and peptide-based phosphorylation enrichment. To increase depth of coverage, both data-dependent and data-independent (via SWATH, Sequential Windowed Acquisition of All Theoretical Fragment Ion Mass Spectra) mass spectrometric acquisitions were performed to obtain a more than 50% increase in coverage of the enriched Chlamydomonas kinome over coverage found with no enrichment. The quantitative phosphoproteomic dataset yielded 2250 phosphopeptides and 1314 localized phosphosites with excellent reproducibility across biological replicates (90% of quantified sites with coefficient of variation below 11%). This approach enables simultaneous investigation of kinases and phosphorylation events at the global level to facilitate understanding of kinase networks and their influence in cell signaling events.

Trophic Mode-Dependent Proteomic Analysis Reveals Functional Significance of Light-Independent Chlorophyll Synthesis in Synechocystis sp. PCC 6803

The photosynthetic model organism Synechocystis sp. PCC 6803 can grow in different trophic modes, depending on the availability of light and exogenous organic carbon source. However, how the protein profile changes to facilitate the cells differentially propagate in different modes has not been comprehensively investigated. Using isobaric labeling-based quantitative proteomics, we simultaneously identified and quantified 45% Synechocystis proteome across four different trophic modes, i.e., autotrophic, heterotrophic, photoheterotrophic, and mixotrophic modes. Among the 155 proteins that are differentially expressed across four trophic modes, proteins involved in nitrogen assimilation and light-independent chlorophyll synthesis are dramatically upregulated in the mixotrophic mode, concomitant with a dramatic increase of PII phosphorylation that senses carbon and nitrogen assimilation status. Moreover, functional study using a mutant defective in light-independent chlorophyll synthesis revealed that this pathway is important for chlorophyll accumulation under a cycled light/dark illumination regime, a condition mimicking day/night cycles in certain natural habitats. Collectively, these results provide the most comprehensive information on trophic mode-dependent protein expression in cyanobacterium, and reveal the functional significance of light-independent chlorophyll synthesis in trophic growth.

Unexpected capacity for organic carbon assimilation by Thermosynechococcus elongatus, a crucial photosynthetic model organism

Genetic modification of key residues of photosystems is essential to identify functionally crucial processes by spectroscopic and crystallographic investigation; the required protein stability favours use of thermophilic species. The currently unique thermophilic photosynthetic model organism is the cyanobacterial genus Thermosynechococcus. We report the ability of Thermosynechococcus elongatus to assimilate organic carbon, specifically D-fructose. Growth in the presence of a photosynthesis inhibitor opens the door towards crucial amino acid substitutions in photosystems by the rescue of otherwise lethal mutations. Yet depression of batch-culture growth after 7 days implies that additional developments are needed.

Detection of outer membrane vesicles in Synechocystis PCC 6803.

It has been well established that many species of Gram-negative bacteria release nanoscale outer membrane vesicles (OMVs) during normal growth. Furthermore, the roles of these structures in heterotrophic bacteria have been extensively characterized. However, little is known about the existence or function of OMVs in photoautotrophs. In the present study, we report for the first time the production of OMVs by the model photosynthetic organism Synechocystis sp. PCC 6803, a species of biotechnological importance. We detected extracellular proteins and lipids in cell-free supernatants derived from Synechocystis culture, yet the cytoplasmic and thylakoid membrane markers NADH oxidase and chlorophyll were absent. This indicated that the extracellular proteins and lipids derived from the outer membrane, and not from cell lysis. Furthermore, we identified spherical structures within the expected size range of OMVs in Synechocystis culture using scanning electron microscopy. Taken together, these results suggest that the repertoire of Gram-negative bacteria that are known to produce OMVs may be expanded to include Synechocystis PCC6803. Because of the considerable genetic characterization of Synechocystis in particular, our discovery has the potential to support novel biotechnological applications as well.

Reduction of PII signaling protein enhances lipid body production in Chlamydomonas reinhardtii

In all examined organisms that have the PII signal transduction machinery, PII coordinates the central C/N anabolic metabolism. In green algae and land plants, PII is localized in the chloroplast and controls the L-arginine biosynthetic pathway pathway. To elucidate additional functions of PII in the model photosynthetic organism Chlamydomonas reinhardtii (CrPII), we generated and analyzed four strains, in which PII was strongly under-expressed by artificial microRNA (GLB1-amiRNA strains). In response to nitrogen deficiency, Chlamydomonas produces triacylglycerols (TAGs) that are accumulated in lipid bodies (LB). Quantification of LBs by confocal microscopy in four GLB1-amiRNA strains showed that reduced PII levels resulted in over-accumulation of LBs compared to their parental strains. Moreover, knock-down of PII caused also an increase in the total TAG level. We propose that the larger yields of TAG-filled LBs in N-starved GLB1-amiRNA cells can be attributed to the strain's depleted PII level and their inability to properly control acetyl-CoA carboxylase activity (ACCase). Together, our results imply that PII in Chlamydomonas negatively controls TAG accumulation in LBs during acclimation to nitrogen starvation of the alga.

Toward the complete proteome of Synechocystis sp. PCC 6803.

The proteome of the photosynthetic model organism Synechocystis sp. PCC6803 has been extensively analyzed in the last 15years for the purpose of identifying proteins specifically expressed in subcellular compartments or differentially expressed in different environmental or internal conditions. This review summarizes the progress achieved so far with the emphasis on the impact of different techniques, both in sample preparation and protein identification, on the increasing coverage of proteome identification. In addition, this review evaluates the current completeness of proteome identification, and provides insights on the potential factors that could affect the complete identification of the Synechocystis proteome.