Iron is a biochemically critical metal cofactor in enzymes involved in photosynthesis, cellular respiration, nitrate assimilation, nitrogen fixation, and reactive oxygen species defense. Marine microeukaryotes have evolved a phytotransferrin-based iron uptake system to cope with iron scarcity, a major factor limiting primary productivity in the global ocean. Diatom phytotransferrin is endocytosed; however, proteins downstream of this environmentally ubiquitous iron receptor are unknown. We applied engineered ascorbate peroxidase APEX2-based subcellular proteomics to catalog proximal proteins of phytotransferrin in the model marine diatom Phaeodactylum tricornutum. Proteins encoded by poorly characterized iron-sensitive genes were identified including three that are expressed from a chromosomal gene cluster. Two of them showed unambiguous colocalization with phytotransferrin adjacent to the chloroplast. Further phylogenetic, domain, and biochemical analyses suggest their involvement in intracellular iron processing. Proximity proteomics holds enormous potential to glean new insights into iron acquisition pathways and beyond in these evolutionarily, ecologically, and biotechnologically important microalgae.
- MeSH
- biologický transport MeSH
- buněčná membrána metabolismus MeSH
- chloroplasty metabolismus MeSH
- multigenová rodina MeSH
- proteomika metody MeSH
- rozsivky genetika metabolismus MeSH
- transferin metabolismus MeSH
- železo metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
Plastids, organelles that evolved from cyanobacteria via endosymbiosis in eukaryotes, provide carbohydrates for the formation of biomass and for mitochondrial energy production to the cell. They generate their own energy in the form of the nucleotide adenosine triphosphate (ATP). However, plastids of non-photosynthetic tissues, or during the dark, depend on external supply of ATP. A dedicated antiporter that exchanges ATP against adenosine diphosphate (ADP) plus inorganic phosphate (Pi) takes over this function in most photosynthetic eukaryotes. Additional forms of such nucleotide transporters (NTTs), with deviating activities, are found in intracellular bacteria, and, surprisingly, also in diatoms, a group of algae that acquired their plastids from other eukaryotes via one (or even several) additional endosymbioses compared to algae with primary plastids and higher plants. In this review, we summarize what is known about the nucleotide synthesis and transport pathways in diatom cells, and discuss the evolutionary implications of the presence of the additional NTTs in diatoms, as well as their applications in biotechnology.
Iron uptake by diatoms is a biochemical process with global biogeochemical implications. In large regions of the surface ocean diatoms are both responsible for the majority of primary production and frequently experiencing iron limitation of growth. The strategies used by these phytoplankton to extract iron from seawater constrain carbon flux into higher trophic levels and sequestration into sediments. In this study we use reverse genetic techniques to target putative iron-acquisition genes in the model pennate diatom Phaeodactylum tricornutum We describe components of a reduction-dependent siderophore acquisition pathway that relies on a bacterial-derived receptor protein and provides a viable alternative to inorganic iron uptake under certain conditions. This form of iron uptake entails a close association between diatoms and siderophore-producing organisms during low-iron conditions. Homologs of these proteins are found distributed across diatom lineages, suggesting the significance of siderophore utilization by diatoms in the marine environment. Evaluation of specific proteins enables us to confirm independent iron-acquisition pathways in diatoms and characterize their preferred substrates. These findings refine our mechanistic understanding of the multiple iron-uptake systems used by diatoms and help us better predict the influence of iron speciation on taxa-specific iron bioavailability.
- MeSH
- biologická dostupnost MeSH
- biologický transport MeSH
- CRISPR-Cas systémy MeSH
- druhová specificita MeSH
- FMN-reduktasa genetika metabolismus MeSH
- fylogeneze MeSH
- galium metabolismus MeSH
- genový knockout MeSH
- klimatické změny MeSH
- membránové transportní proteiny genetika metabolismus MeSH
- mikrobiota MeSH
- mořská voda chemie MeSH
- oxidace-redukce MeSH
- proteiny vnější bakteriální membrány metabolismus MeSH
- receptory buněčného povrchu metabolismus MeSH
- rekombinantní fúzní proteiny metabolismus MeSH
- rozsivky genetika růst a vývoj metabolismus MeSH
- siderofory metabolismus MeSH
- železo metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
Diatoms are unicellular algae and evolved by secondary endosymbiosis, a process in which a red alga-like eukaryote was engulfed by a heterotrophic eukaryotic cell. This gave rise to plastids of remarkable complex architecture and ultrastructure that require elaborate protein importing, trafficking, signaling and intracellular cross-talk pathways. Studying both plastids and mitochondria and their distinctive physiological pathways in organello may greatly contribute to our understanding of photosynthesis, mitochondrial respiration and diatom evolution. The isolation of such complex organelles, however, is still demanding, and existing protocols are either limited to a few species (for plastids) or have not been reported for diatoms so far (for mitochondria). In this work, we present the first isolation protocol for mitochondria from the model diatom Thalassiosira pseudonana. Apart from that, we extended the protocol so that it is also applicable for the purification of a high-quality plastids fraction, and provide detailed structural and physiological characterizations of the resulting organelles. Isolated mitochondria were structurally intact, showed clear evidence of mitochondrial respiration, but the fractions still contained residual cell fragments. In contrast, plastid isolates were virtually free of cellular contaminants, featured structurally preserved thylakoids performing electron transport, but lost most of their stromal components as concluded from Western blots and mass spectrometry. Liquid chromatography electrospray-ionization mass spectrometry studies on mitochondria and thylakoids, moreover, allowed detailed proteome analyses which resulted in extensive proteome maps for both plastids and mitochondria thus helping us to broaden our understanding of organelle metabolism and functionality in diatoms.
Diatoms outcompete other phytoplankton for nitrate, yet little is known about the mechanisms underpinning this ability. Genomes and genome-enabled studies have shown that diatoms possess unique features of nitrogen metabolism however, the implications for nutrient utilization and growth are poorly understood. Using a combination of transcriptomics, proteomics, metabolomics, fluxomics, and flux balance analysis to examine short-term shifts in nitrogen utilization in the model pennate diatom in Phaeodactylum tricornutum, we obtained a systems-level understanding of assimilation and intracellular distribution of nitrogen. Chloroplasts and mitochondria are energetically integrated at the critical intersection of carbon and nitrogen metabolism in diatoms. Pathways involved in this integration are organelle-localized GS-GOGAT cycles, aspartate and alanine systems for amino moiety exchange, and a split-organelle arginine biosynthesis pathway that clarifies the role of the diatom urea cycle. This unique configuration allows diatoms to efficiently adjust to changing nitrogen status, conferring an ecological advantage over other phytoplankton taxa.
- MeSH
- biologické modely MeSH
- chloroplasty genetika metabolismus MeSH
- dusičnany metabolismus MeSH
- dusík metabolismus MeSH
- metabolické sítě a dráhy genetika MeSH
- metabolomika metody MeSH
- mitochondrie genetika metabolismus MeSH
- molekulární evoluce MeSH
- mořská voda mikrobiologie MeSH
- proteomika metody MeSH
- regulace genové exprese MeSH
- rozsivky genetika metabolismus MeSH
- signální transdukce genetika MeSH
- stanovení celkové genové exprese metody MeSH
- uhlík metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
The establishment of the mitochondrion is seen as a transformational step in the origin of eukaryotes. With the mitochondrion came bioenergetic freedom to explore novel evolutionary space leading to the eukaryotic radiation known today. The tight integration of the bacterial endosymbiont with its archaeal host was accompanied by a massive endosymbiotic gene transfer resulting in a small mitochondrial genome which is just a ghost of the original incoming bacterial genome. This endosymbiotic gene transfer resulted in the loss of many genes, both from the bacterial symbiont as well the archaeal host. Loss of genes encoding redundant functions resulted in a replacement of the bulk of the host's metabolism for those originating from the endosymbiont. Glycolysis is one such metabolic pathway in which the original archaeal enzymes have been replaced by bacterial enzymes from the endosymbiont. Glycolysis is a major catabolic pathway that provides cellular energy from the breakdown of glucose. The glycolytic pathway of eukaryotes appears to be bacterial in origin, and in well-studied model eukaryotes it takes place in the cytosol. In contrast, here we demonstrate that the latter stages of glycolysis take place in the mitochondria of stramenopiles, a diverse and ecologically important lineage of eukaryotes. Although our work is based on a limited sample of stramenopiles, it leaves open the possibility that the mitochondrial targeting of glycolytic enzymes in stramenopiles might represent the ancestral state for eukaryotes.
- MeSH
- biologická evoluce MeSH
- Blastocystis cytologie enzymologie genetika metabolismus MeSH
- energetický metabolismus MeSH
- genom mitochondriální MeSH
- glykolýza * MeSH
- mitochondrie genetika metabolismus MeSH
- rozsivky cytologie enzymologie genetika metabolismus MeSH
- symbióza MeSH
- transformace genetická MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Strong excitonic interactions are a key design strategy in photosynthetic light harvesting, expanding the spectral cross-section for light absorption and creating considerably faster and more robust excitation energy transfer. These molecular excitons are a direct result of exceptionally densely packed pigments in photosynthetic proteins. The main light-harvesting complexes of diatoms, known as fucoxanthin-chlorophyll proteins (FCPs), are an exception, displaying surprisingly weak excitonic coupling between their chlorophyll (Chl) a's, despite a high pigment density. Here, we show, using single-molecule spectroscopy, that the FCP complexes of Cyclotella meneghiniana switch frequently into stable, strongly emissive states shifted 4-10 nm toward the red. A few percent of isolated FCPa complexes and ∼20% of isolated FCPb complexes, on average, were observed to populate these previously unobserved states, percentages that agree with the steady-state fluorescence spectra of FCP ensembles. Thus, the complexes use their enhanced sensitivity to static disorder to increase their light-harvesting capability in a number of ways. A disordered exciton model based on the structure of the main plant light-harvesting complex explains the red-shifted emission by strong localization of the excitation energy on a single Chl a pigment in the terminal emitter domain due to very specific pigment orientations. We suggest that the specific construction of FCP gives the complex a unique strategy to ensure that its light-harvesting function remains robust in the fluctuating protein environment despite limited excitonic interactions.
In the present work, we report the first comparative spectroscopic investigation between Photosystem I (PSI) complexes isolated from two red clade algae. Excitation energy transfer was measured in PSI from Chromera velia, an alga possessing a split PsaA protein, and from the model diatom Phaeodactylum tricornutum. In both cases, the estimated effective photochemical trapping time was in the 15-25ps range, i.e. twice as fast as higher plants. In contrast to green phototrophs, the trapping time was rather constant across the whole emission spectrum. The weak wavelength dependence was attributed to the limited presence of long-wavelength emitting chlorophylls, as verified by low temperature spectroscopy. As the trapping kinetics of C. velia PSI were barely distinguishable from those of P. tricornutum PSI, it was concluded that the scission of PsaA protein had no significant impact on the overall PSI functionality. In conclusion, the two red clade algae analysed here, carried amongst the most efficient charge separation so far reported for isolated Photosystems.
- MeSH
- Alveolata metabolismus MeSH
- chlorofyl metabolismus MeSH
- fluorescenční spektrometrie MeSH
- fotosystém I - proteinový komplex metabolismus MeSH
- kinetika MeSH
- přenos energie fyziologie MeSH
- Rhodophyta metabolismus MeSH
- rozsivky metabolismus MeSH
- světlosběrné proteinové komplexy metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Tetrapyrroles such as chlorophyll and heme are indispensable for life because they are involved in energy fixation and consumption, i.e. photosynthesis and oxidative phosphorylation. In eukaryotes, the tetrapyrrole biosynthetic pathway is shaped by past endosymbioses. We investigated the origins and predicted locations of the enzymes of the heme pathway in the chlorarachniophyte Bigelowiella natans, the cryptophyte Guillardia theta, the "green" dinoflagellate Lepidodinium chlorophorum, and three dinoflagellates with diatom endosymbionts ("dinotoms"): Durinskia baltica, Glenodinium foliaceum and Kryptoperidinium foliaceum. Bigelowiella natans appears to contain two separate heme pathways analogous to those found in Euglena gracilis; one is predicted to be mitochondrial-cytosolic, while the second is predicted to be plastid-located. In the remaining algae, only plastid-type tetrapyrrole synthesis is present, with a single remnant of the mitochondrial-cytosolic pathway, a ferrochelatase of G. theta putatively located in the mitochondrion. The green dinoflagellate contains a single pathway composed of mostly rhodophyte-origin enzymes, and the dinotoms hold two heme pathways of apparently plastidal origin. We suggest that heme pathway enzymes in B. natans and L. chlorophorum share a predominantly rhodophytic origin. This implies the ancient presence of a rhodophyte-derived plastid in the chlorarachniophyte alga, analogous to the green dinoflagellate, or an exceptionally massive horizontal gene transfer.
- MeSH
- biologická evoluce * MeSH
- biosyntetické dráhy * genetika MeSH
- Cryptophyta klasifikace genetika metabolismus MeSH
- Dinoflagellata klasifikace genetika metabolismus MeSH
- fylogeneze MeSH
- hem metabolismus MeSH
- porfobilinogensynthasa genetika metabolismus MeSH
- rozsivky klasifikace genetika metabolismus MeSH
- stanovení celkové genové exprese MeSH
- tetrapyrroly metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
