Transcriptional response of the marine pennate diatom Phaeodactylum tricornutum to Fe limitation
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ABSTRACT: Diatoms, which are responsible for up to 40% of the 45 to 50 billion metric tons of organic carbon production each year in the sea, are particularly sensitive to Fe stress. Here we describe the transcriptional response of the pennate diatom Phaeodactylum tricornutum to Fe limitation using a partial genome microarray based on EST and genome sequence data. Processes carried out by components rich in Fe, such as photosynthesis, mitochondrial electron transport and nitrate assimilation are down-regulated to cope with the reduced cellular iron quota. This retrenchment is compensated by nitrogen (N) and carbon (C) reallocation from protein and storage carbohydrate degradation, adaptations to chlorophyll biosynthesis and pigment metabolism, removal of excess electron s by mitochondrial alternative oxidase (AOX), augmented Fe-independent oxidative stress responses, and sensitized iron capture mechanisms. Keywords: Marine phytoplankton, pinnate diatom
Project description:Diatoms, which are responsible for up to 40% of the 45 to 50 billion metric tons of organic carbon production each year in the sea, are particularly sensitive to Fe stress. Here we describe the transcriptional response of the pennate diatom Phaeodactylum tricornutum to Fe limitation using a partial genome microarray based on EST and genome sequence data. Processes carried out by components rich in Fe, such as photosynthesis, mitochondrial electron transport and nitrate assimilation are down-regulated to cope with the reduced cellular iron quota. This retrenchment is compensated by nitrogen (N) and carbon (C) reallocation from protein and storage carbohydrate degradation, adaptations to chlorophyll biosynthesis and pigment metabolism, removal of excess electron s by mitochondrial alternative oxidase (AOX), augmented Fe-independent oxidative stress responses, and sensitized iron capture mechanisms. Keywords: Marine phytoplankton, pinnate diatom Wild-type Phaeodactylum tricornutum was grown under Fe replete (10,000 nM) and Fe limiting (5nM) conditions. Partial genome gene expression analysis of iron-inducible genes was conducted using a two-color competitive hybridization microarray.
Project description:Iron and manganese are part of a small group of transition metals required for photosynthetic electron transport. Here, we present evidence for a functional link between iron and manganese homeostasis. In the unicellular cyanobacterium, Synechocystis sp. PCC 6803 Fe and Mn deprivation resulted in distinct modifications of the function of the photosynthetic apparatus. For example, iron limitation modifies the rate of QA re-oxidation in photosystem II, a complex that contains more Mn than Fe. The intracellular elemental quotas of Fe and Mn are also linked. Fe limitation reduces the intracellular Mn quota. Mn limitation did not exert a reciprocal effect on Fe quotas. Microarray analysis comparing Mn and Fe limitation revealed a stark difference in the extent of the transcriptional response to the two limiting conditions, reflective of the physiological data. The effects of Fe limitation on the transcriptional network are widespread while the effects on Mn limitation are highly specific. Our analysis also revealed an overlap in the transcriptional response of specific Fe and Mn transporters. This overlap provides a framework for explaining Fe limitation induced changes in Mn quotas. Fe transporters can serve as a low affinity Mn transport system. Under iron limitation the specificity of the Fe transport system changes, making it a less efficient Mn transport system.
Project description:Iron and manganese are part of a small group of transition metals required for photosynthetic electron transport. Here, we present evidence for a functional link between iron and manganese homeostasis. In the unicellular cyanobacterium, Synechocystis sp. PCC 6803 Fe and Mn deprivation resulted in distinct modifications of the function of the photosynthetic apparatus. For example, iron limitation modifies the rate of QA re-oxidation in photosystem II, a complex that contains more Mn than Fe. The intracellular elemental quotas of Fe and Mn are also linked. Fe limitation reduces the intracellular Mn quota. Mn limitation did not exert a reciprocal effect on Fe quotas. Microarray analysis comparing Mn and Fe limitation revealed a stark difference in the extent of the transcriptional response to the two limiting conditions, reflective of the physiological data. The effects of Fe limitation on the transcriptional network are widespread while the effects on Mn limitation are highly specific. Our analysis also revealed an overlap in the transcriptional response of specific Fe and Mn transporters. This overlap provides a framework for explaining Fe limitation induced changes in Mn quotas. Fe transporters can serve as a low affinity Mn transport system. Under iron limitation the specificity of the Fe transport system changes, making it a less efficient Mn transport system. We monitored the gene expression of Synechocystis PCC6083 at standard conditions and after 2 days of iron limitation (0Fe), manganese limitation (0Mn) and combined iron and manganese limitation (0Fe0Mn). Each timepoint and condition was sampled in triplicates. Due to strong deviations in one of the three repeats for the 0Mn and 0Fe0Mn conditions, the corresponding replicates were excluded from further analysis.
Project description: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-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 Fe-limited stress, diatoms alter plastid-specific processes, including components of electron transport. These physiological changes suggest changes of protein content and their abundance 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 model photosynthetic organisms. To characterize proteins enriched in diatom plastids we have used shotgun proteomics to assess the proteome of subcellular plastid-enriched fractions from Thalassiosira pseudonana. To improve our understanding of how the plastid proteome is remodeled in response to Fe limitation, proteome sequencing has been performed on T. pseudonana grown under Fe replete and limited conditions. These analyses have shown that Fe limitation regulates major metabolic pathways in the plastid, including the Calvin cycle, as well as changes in light harvesting protein expression. In-silico localization predictions of proteins identified in this plastid-enriched proteome allowed for an in-depth comparison of theoretical vs observed plastid-localization, providing evidence for the potential of additional protein import pathways into the diatom plastid.
Project description:Geobacter species are of great interest for environmental and biotechnology applications as they can carry out direct electron transfer to insoluble metals or other microorganisms and have the ability to assimilate inorganic carbon. Here, we report on the capability and key enabling metabolic machinery of Geobacter metallireducens GS-15 to carry out CO2 fixation and direct electron transfer to iron. An updated metabolic reconstruction was generated, growth screens on targeted conditions of interest were performed, and constraint-based analysis was utilized to characterize and evaluate critical pathways and reactions in G. metallireducens. The novel capability of G. metallireducens to grow autotrophically with formate and Fe(III) was predicted and subsequently validated in vivo. Additionally, the energetic cost of transferring electrons to an external electron acceptor was determined through analysis of growth experiments carried out using three different electron acceptors (Fe(III), nitrate, and fumarate) by systematically isolating and examining different parts of the electron transport chain. The updated reconstruction will serve as a knowledgebase for understanding and engineering Geobacter and similar species.
Project description:Here, we examined the ramifications of between-species diversity by documenting the transcriptional response of three marine diatoms - Thalassiosira pseudonana, Fragilariopsis cylindrus, and Pseudo-nitzschia multiseries - to the onset of nitrate limitation of growth, a common limiting nutrient in the ocean. Less than 5% of orthologous genes, shared across the three diatoms, displayed the same transcriptional responses across species when growth was limited by nitrate availability. Orthologs, such as those involved in nitrogen uptake and assimilation, as well as carbon metabolism, were differently expressed across the three species. The two pennate diatoms, F. cylindrus and P. multiseries, shared 3,839 clusters without orthologs in the genome of the centric diatom T. pseudonana. A majority of these pennate-clustered genes, as well as the non-orthologous genes in each species, had minimal annotation information, but were often significantly differentially expressed under nitrate limitation, indicating their potential importance in the response to nitrogen availability. Despite these variations in the specific transcriptional response of each diatom, overall transcriptional patterns suggested that all three diatoms displayed a common physiological response to nitrate limitation that consisted of a general reduction in carbon fixation and carbohydrate and fatty acid metabolism and an increase in nitrogen recycling.
Project description:Diatoms are prominent marine microalgae, interesting not only from an ecological point of view, but also for their possible use for biotechnology applications. They can be cultivated in phototrophic conditions, using sunlight as the only energy source. Some diatoms, however, can also grow in mixotrophic mode, where both light and external reduced carbon contribute to biomass accumulation. In this study, we investigated the consequences of mixotrophy on the growth and metabolism of the pennate diatom Phaeodactylum tricornutum, using glycerol as a source of reduced carbon. Transcriptomic, metabolomic and physiological data indicate that glycerol affects the central-carbon, carbon-storage and lipid metabolism of the diatom. In particular, glycerol addition mimics some typical responses of nitrogen limitation on lipid metabolism at the level of TAG accumulation and fatty acid composition. However, this compound does not diminish photosynthetic activity and cell growth, at variance with nutrient limitation, revealing essential aspects of the metabolic flexibility of these microalgae and suggesting possible biotechnological applications of mixotrophy.
Project description:Low iron (Fe) bioavailability can limit the biosynthesis of Fe-containing proteins, which are especially abundant in photosynthetic organisms, thus negatively affecting global primary productivity. Understanding cellular coping mechanisms under Fe limitation is therefore of great interest. We surveyed the temporal responses of Chlamydomonas (Chlamydomonas reinhardtii) cells transitioning from an Fe-rich to an Fe-free medium to document their short- and long-term adjustments. While slower growth, chlorosis and lower photosynthetic parameters are evident only after one or more days in Fe-free medium, the abundance of some transcripts, such as those for genes encoding transporters and enzymes involved in Fe assimilation, change within minutes, before changes in intracellular Fe content are noticeable, suggestive of a sensitive mechanism for sensing Fe. Promoter reporter constructs indicate a transcriptional component to this immediate primary response. With acetate provided as a source of reduced carbon, transcripts encoding respiratory components are maintained relative to transcripts encoding components of photosynthesis and tetrapyrrole biosynthesis, indicating metabolic prioritization of respiration over photosynthesis. In contrast to the loss of chlorophyll, carotenoid content is maintained under Fe limitation despite a decrease in the transcripts for carotenoid biosynthesis genes, indicating carotenoid stability. These changes occur more slowly, only after the intracellular Fe quota responds, indicating a phased response in Chlamydomonas, involving both primary and secondary responses during acclimation to poor Fe nutrition.
Project description:Here, we examined the ramifications of between-species diversity by documenting the transcriptional response of three marine diatoms - Thalassiosira pseudonana, Fragilariopsis cylindrus, and Pseudo-nitzschia multiseries - to the onset of nitrate limitation of growth, a common limiting nutrient in the ocean. Less than 5% of orthologous genes, shared across the three diatoms, displayed the same transcriptional responses across species when growth was limited by nitrate availability. Orthologs, such as those involved in nitrogen uptake and assimilation, as well as carbon metabolism, were differently expressed across the three species. The two pennate diatoms, F. cylindrus and P. multiseries, shared 3,839 clusters without orthologs in the genome of the centric diatom T. pseudonana. A majority of these pennate-clustered genes, as well as the non-orthologous genes in each species, had minimal annotation information, but were often significantly differentially expressed under nitrate limitation, indicating their potential importance in the response to nitrogen availability. Despite these variations in the specific transcriptional response of each diatom, overall transcriptional patterns suggested that all three diatoms displayed a common physiological response to nitrate limitation that consisted of a general reduction in carbon fixation and carbohydrate and fatty acid metabolism and an increase in nitrogen recycling. Transcriptomes were collected for diatom cultures harvested at the onset of stationary phase in low nitrate media (55 M-NM-<M NaNO3, 212 M-NM-<M Na2SiO3, 72.4 M-NM-<M NaH2PO4) or during mid-exponential growth in nutrient-replete media (882 M-NM-<M NaNO3, 106 M-NM-<M Na2SiO3, 36.2 M-NM-<M NaH2PO4) in artificial seawater, maintaining three biological replicates per condition and per diatom (N=18). The SOLiD sequencer (version 4) was used to generate the transcriptomes and the SEAStAR software package was used to process the SOLiD reads and to calculate gene counts. Pooled counts for the nitrate-limited treatment were normalized to pooled counts for the nutrient-replete M-bM-^@M-^\controlM-bM-^@M-^] treatment to generate log fold changes in gene transcription using the R software package edgeR from Bioconductor.